Time-delay integration in a flow cytometry system

ABSTRACT

An apparatus for detecting analytes in a sample is provided. The apparatus can include: a flow cytometry system including one or more detection zones; one or more irradiation sources disposed for irradiating the one or more detection zones with radiation; at least one detector disposed for collecting charges corresponding to light signals emitted from detectable analytes in the one or more detection zones, which have been excited by the radiation, wherein at least one detector includes an output; a system coupled to the at least one detector for effecting time delay integration of the charges by accumulating the charges before reading the charges at the output of the at least one detector; and a sorting mechanism, for example, to effect flow cytometry. Methods for detecting analytes in a sample are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/205,028 filed Jul. 25, 2002, which claims a benefit fromU.S. Provisional Application No. 60/307,682 filed Jul. 25, 2001, andthis application also claims benefit under 35 U.S.C. Section 119(e) fromearlier U.S. Provisional Patent Application No. 60/485,468, filed Jul.8, 2003, and this application also claims benefit under 35 U.S.C.Section 119(e) from earlier U.S. Provisional Patent Application No.60/486,112, filed Jul. 10, 2003, all of which are incorporated herein intheir entireties by reference. U.S. patent application No. Ser.10/805,096, filed Mar. 19, 2004, to Reel et al., is also incorporatedherein in its entirety by reference.

FIELD

The present teachings relate to a detection method useful in a flowcytometry system.

BACKGROUND

Well-known examples of biopolymer analysis using DNA sequencing aretaught, for example, in F. Sanger et al., DNA Sequencing with ChainTerminating Inhibitors, 74 Proc. Nat. Acad. Sci. USA 5463 (1977); LloydM. Smith et al., Fluorescence detection in automated DNA sequenceanalysis, 321 Nature 674 (1986); and Lloyd M. Smith, The Future of DNASequencing, 262 Science 530 (1993). These and all other publications andpatents cited herein are incorporated herein in their entireties byreference.

The use of sources of irradiation other than lasers for the excitationof marker compounds provides many advantages. Although the use of lightemitting diodes (LEDs) for generating fluorescence in dye molecules istaught, for example, in U.S. Pat. Nos. 6,005,663 and 5,710,628, thecontents of which are incorporated herein in their entireties byreference, the use of LEDs in such electrophoretic methods typicallyresults in low signal strengths and marginal detection sensitivity. Thelow signal strength can impair adequate detection of marker compounds.

An electrophoretic and/or other separation apparatus and method thatincludes a cost-effective and convenient source of irradiation and thatdoes not compromise sensitivity or resolution would be desirable,especially in a multiple-channel electrophoretic or flow cytometrysystem.

SUMMARY

According to various embodiments, an apparatus for detecting analytes ina sample containing at least one analyte is provided. The apparatus caninclude: a flow cytometry system including a channel having one or moredetection zones; one or more irradiation sources disposed forirradiating the one or more detection zones with non-coherent radiation;at least one detector disposed for collecting at least one chargecorresponding to an emission beam emitted from the one or more detectionzones, the at least one detector having at least one output; modulatingoptics disposed between the irradiation source and the at least onedetector; and a time delay integration system coupled to the at leastone detector for effecting time delay integration of the at least onecharge by accumulating the at least one charge before reading the atleast one charge at the output of the at least one detector. The timedelay integration system can accumulate the at least one charge bymoving, relative to one another, the modulating optics and the channel.

According to various embodiments, an apparatus for detecting analytes ina sample is provided. The apparatus can include: a flow cytometry systemincluding a channel having at least one detection zone; one or moreirradiation sources disposed for irradiating the at least one detectionzone with radiation; at least one detector disposed for collecting atleast one charge corresponding to an emission beam emitted from the atleast one detection zone, each detector of the at least one detectorhaving an output; modulating optics disposed between the irradiationsource and the at least one detector; and a time delay integrationsystem coupled to the at least one detector for effecting time delayintegration of the at least one charge by accumulating the at least onecharge before reading the at least one charge at the output of the atleast one detector. The time delay integration system can accumulate theat least one charge by moving, relative to one another, the modulatingoptics and the channel. The one or more irradiation sources can includea solid state laser or a micro-wire laser.

According to various embodiments, an apparatus for sorting analytes in asample containing at least one detectable analyte is provided. Theapparatus can include: a channel having one or more detection zones; oneor more irradiation sources disposed for irradiating the one or moredetection zones with non-coherent radiation; at least one detectordisposed for collecting light signals emitted from the at least onedetectable analyte in the one or more detection zones excited by theradiation, the at least one detector having an output; a time delayintegration system coupled to the at least one detector for effectingthe time delay integration of at least one charge on the at least onedetector, corresponding to the light signals by accumulating the atleast one charge before reading the at least one charge at the output ofthe at least one detector; and a sorting system capable of directing theflow of at least one detectable analyte.

According to various embodiments, a method for sorting analytes in asample containing at least one detectable analyte is provided. Themethod can include providing a channel-defining-member defining achannel therein having at least one detection zone. The method caninclude separating a sample containing at least one detectable analytemoving through the channel. The method can include irradiating the atleast detection zone using one or more irradiation sources generatingradiation of such wavelength as to thereby excite the at least onedetectable analyte and cause the at least one detectable analyte to emitlight signals indicative of the at least one detectable analyte. Themethod can include detecting the light signals produced by the at leastone detectable analyte by collecting the light signals on at least onedetector to produce charges on the at least one detector correspondingto the light signals. The method can include modulating light betweenthe one or more irradiation sources and the at least one detector usingmodulating optics. The method can include effecting a time delayintegration of the light signals within the at least one detector byaccumulating at least one charge within the at least one detectorcorresponding to light signals associated with the at least onedetectable analyte during an integration time of the at least onedetectable analyte moving across the at least one detection zone. Theaccumulation can be effected by moving, relative to one another, themodulating optics and the channel. The method can include reading the atleast one accumulated charge. The method can include sorting adetectable analyte of the sample based on the reading of the at leastone accumulated charge. The sorting can be performed with a flowcytometry apparatus and method, for example.

According to various embodiments, an apparatus is provided that caninclude a sorting system for separating each of a plurality of detectedanalytes into respective collections of analytes, and which uses atime-delay integration detection system to detect the analytes. Thesorting system can include a motive force for directing the analyte toan analyte collector. The collector can include a dish, channel,capillary tube, beaker, or other device capable of retaining theanalyte. The motive force can act on the analyte, on the collector, orboth. The motive force can be an electro-kinetic force, a mechanicalforce, an electric field gradient, a vacuum, or a combination thereof.

According to various embodiments, the apparatus can include a separationdevice for directing a detected component to flow along one or morepathways, for example, by electrokinetic movement or mechanical movementof the detected component or by such movement of the pathway forreceiving the detected component. The apparatus can further include asystem coupled to the at least one detector for effecting time delayintegration of the charges on the at least one detector corresponding tothe light signals by accumulating the charges before reading the chargesat the output of the at least one detector.

According to various embodiments, the flow cytometry system can includean electric field gradient source. The one or more irradiation sourcescan include one or more light emitting diodes. The one or more lightemitting diodes can include one or more organic light emitting diodes.The modulating optics can include a relay lens system comprising acollimating lens and a re-imaging lens. The modulating optics caninclude a conditioning filter disposed between the one or moreirradiation sources and the one or more detection zones. Theconditioning filter can include a longpass filter, a shortpass filter, amulti-notch filter, a beamsplitter, or a combination thereof. Themodulating optics can include a focusing lens disposed between theconditioning filter and the one or more detection zones. The modulatingoptics can include a transmission grating disposed between the focusinglens and the re-imaging lens. The modulating optics can include a filterdisposed between the one or more detection zones and the at least onedetector for filtering through only the emission beam. The filter caninclude a longpass filter, a shortpass filter, a multi-notch filter, abandpass filter, a beam splitter, or a combination thereof.

According to various embodiments, the apparatus can include a samplecontaining whole cells. The appartus can include a sample containing theat least one analyte labeled with at least one marker. The at least onemarker can include a dye marker, a fluorescing dye, a free-floating dye,a reporter dye, a probe dye, an intercalating dye, a quantum dot, amolecular beacon, a quantum dot media, a quantum dot bead, a dye-labeledbead, a dye attached to an analyte assoicated with a bead, or acombination thereof. The modulating optics can include at least oneconditioning filter for each irradiation source of the one or moreirradiation sources, each respective conditioning filter being effectivefor substantially blocking predetermined excitation wavelengths toproduce conditioned light. The predetermined excitation wavelengths ofeach marker can be without conflict with the excitation spectra of eachother marker. The modulating optics can include at least one long passfilter and at least one bandpass filter for each irradiation source ofthe one or more irradiation sources, each of the at least one long passfilter and each of the at least one bandpass filter being effective forletting through, substantially exclusively, predetermined wavelengths oflight from the one or more detection zones corresponding to a portion ofthe wavelengths of the light signals emitted by the at least one marker,to thereby produce filtered light.

According to various embodiments, the apparatus can include a seperatingdevice. The seperating device can include a plurality of separationregions. The one or more irradiation sources can include a single lightemitting diode. The apparatus can include a device capable of spectrallydistributing the light signals to thereby produce spectrally distributedlight. The one or more irradiation sources can include a plurality oflight emitting diodes each emitting light in a respective predeterminedfrequency range, and the respective bandpass filter can include aplurality of bandpass filters each associated with a respective one ofthe plurality of light emitting diodes, each respective bandpass filterbeing effective for letting through, substantially exclusively,predetermined wavelengths of light from the one or more detection zonescorresponding to a portion of the wavelengths of the light signalsemitted by each marker of the at least one marker to thereby producefiltered light.

According to various embodiments, the apparatus can include an offsetsystem for spatially offsetting, on the at least one detector, at leastone image for each conditioning filter by a predetermined amount. Theoffset system can include a plurality of offset mechanisms, eachassociated with a respective one of the conditioning filters. Eachoffset mechanism can include one or more of a glass plate, a grating, amirror, or a combination thereof. The offset system can be adapted toeffect a translational movement of at least one of the at least onedetector, the modulating optics, and the one or more detection zones,with respect to one another, for spatially offsetting the at least oneimage by a second predetermined amount.

According to various embodiments, the apparatus can include a filterwheel. The bandpass filters can be disposed on the filter wheel, witheach respective bandpass filter further being selectively positionablewith respect to the one or more detection zones for filtering lightemitted from the one or more detection zones by the at least one markerassociated with each respective bandpass filter.

According to various embodiments, the time delay integration system cancontrol the at least one detector to read the at least one accumulatedcharge on a frame by frame basis, each frame corresponding to the atleast one accumulated charge on the at least one detector during anintegration time and produced by the conditioned light through eachconditioning filter. The time delay integration system can control theat least one detector to read the at least one accumulated charge on acontinuous basis. The at least one detector can include atwo-dimensional charge-coupled device. The one or more irradiationsources can include a plurality of light emitting diodes adapted tosimultaneously irradiate the one or more detection zones, each of thelight emitting diodes illuminating a separate one of the one or moredetection zones. The apparatus can include masks to selectively mask thechannel such that the light signals from the respective one or moredetection zones can be distinct. The time delay integration system caninclude a system coupled to the modulating optics for moving themodulating optics relative to the channel at a speed that issynchronized to a movement of the at least one analyte across the one ormore detection zones. The time delay integration system can include asystem coupled to the channel for moving the channel relative to themodulating optics at a speed that is synchronized to a movement of theat least one analyte across the one or more detection zones.

According to various embodiments, the apparatus can include a sortingsystem including one or more collection channels and a controller forcontrolling a motive force for each collection channel. The sortingsystem can sort each analyte into a respective one of the one or morecollection channels. The motive force can include an electrokineticforce, a mechanical force, a production of electric field gradient, aswitchable electric field, vacuum, a stream of air, or a combinationthereof. The controller can include more than one controller, eachcontroller associated with a respective collection channel.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only.The accompanying drawings, which are incorporated in and constitute apart of this application, illustrate several exemplary embodiments and,together with the instant description, serve to explain the principlesof the present teachings.

BRIEF DESCRIPTION OF DRAWINGS

Various embodiments of the present teachings are exemplified in theaccompanying drawings. The teachings are not limited to the embodimentsdepicted, and include equivalent structures and methods as set forth inthe following description and known to those of ordinary skill in theart. In the drawings:

FIG. 1 is a schematic, side-elevational view of an electrophoresisarrangement showing a channel-defining member in cross-section,according to various embodiments;

FIG. 2 is a schematic view of an image produced on a detector array of adetector at a time t using the arrangement of FIG. 1;

FIG. 3 is a view similar to FIG. 2 showing the image at a time t+Δt;

FIG. 4 is a schematic, front-elevational view of an electrophoresisarrangement for the sequential use of multiple-color irradiation sourcesalong with filters on a filter wheel, according to various embodiments;

FIG. 5 a is a schematic, top-plan view of the arrangement of FIG. 4;

FIG. 5 b is a schematic, side-elevational view of the arrangement ofFIG. 4;

FIGS. 6 a through 6 e are respective schematic views of images producedon the detector array of a detector, each image corresponding to lightsignals filtered through a respective filter on the filter wheel of FIG.4;

FIG. 6 f is a schematic representation of an electropherogram showingfluorescence intensity curves for each filter of the filter wheel inFIG. 4 during three signal readings by the detector;

FIG. 6 g is a schematic representation of the intensity curves of FIG. 6f in aligned format for multicomponenting;

FIG. 6 h is a schematic representation of multicomponented intensitycurves for five different kinds of markers that can be used in thesystem of FIG. 4 based on the readings in FIG. 6 f;

FIG. 6 i is a graph of excitation efficiency versus wavelength for fourexemplary markers;

FIG. 6 j is a graph of fluorescence intensity versus wavelength for theexemplary markers of FIG. 6 i;

FIG. 7 is a top-plan, partially cross-sectional view, of anelectrophoresis arrangement for the sequential use of multiple colorirradiation sources along with filters on a filter wheel and along withan offset system, according to various embodiments;

FIG. 8 is a schematic view of an image produced on the detector array ofa detector using the arrangement of FIG. 7;

FIG. 9 a is a graph of relative excitation intensity versus wavelengthfor a pair of LEDs used in another embodiment, the LEDs can be ofdifferent colors and can be used to irradiate the detection zonesimultaneously;

FIG. 9 b is a graph showing percent transmission versus wavelength for aconditioning filter used to condition the light from the LEDs of FIG. 9a;

FIG. 9 c is a graph showing percent transmission versus wavelength for abandpass filter used to filter light signals produced by markers excitedby the light from the LEDs of FIG. 9 a;

FIG. 9 d is a graph showing relative emission intensity versuswavelength for the light filtered through the bandpass filter of FIG. 9c;

FIG. 10 is a schematic, top-plan view of an irradiation zone showingthree channels having been selectively masked to present respectivewindows according to another embodiment;

FIG. 11 is a schematic view of a detector array, the detector arrayhaving been separated into respective frames for use with light signalsemitted from the respective windows of the channels in FIG. 10;

FIG. 12 a is a partially cut away view of one of the frames of thedetector array shown in FIG. 11;

FIG. 12 b is a graph showing percent integration per pixel throughoutthe width of the frame as shown in FIG. 12 a;

FIG. 13 a is a schematic of a portion of an electrophoresis arrangementshowing electrostatic sorting of the analytes or components, accordingto various embodiments;

FIG. 13 b is a schematic of a portion of an electrophoresis arrangementshowing mechanical sorting of the analytes or components, according tovarious embodiments; and

FIG. 13 c is a schematic of a portion of an electrophoresis arrangementshowing sorting of the analytes or components into various channels of amulti-channel microcard, according to various embodiments.

FIG. 14 illustrates an exemplary embodiment of a light source layout,for example, an organic light emitting diode (OLED) layout with varyingcolor OLEDs stacked upon each other.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide a further explanation of the variousembodiments of the present teachings.

DESCRIPTION

The analytes of interest can be detected on the basis of anintrinsically detectable signal, or may be derivatized with a labelwhich confers a desired type of detectability. Various methods forlabeling analytes with detectable moieties are well known in the art,such as excitable reporters, radioactive isotopes, fluorescent dyes,spin labels, chemiluminescent compounds and the like to stimulatedetectable emission indicative of the nature of the analyte. When theanalyte is a polynucleotide, labeling by hybridization with a labeledprobe can also be used. The analyte can be labeled with a dye marker, afluorescing dye, a free-floating dye, a reporter dye, a probe dye, anintercalating dye, a quantum dot, a molecular beacon, a linear probe, aquantum dot media, a quantum dot bead, a dye-labeled bead, a dyeattached to an analyte associated with a bead, or a combination thereof.

While the present teachings are related more generally to separationdevices including a time-delay integration detection capability ormethod, the teachings can be exemplified with reference to theelectrophoretic separation system described herein with reference toFIGS. 1-12 b. Although the detectable components described herein arereferred to as analytes or analyte bands, it should be noted that suchanalytes can include whole cells, whole blood cells, nucleic acidsequences, and other biological samples.

Exemplary analytes can include nucleic acids, both single and doublestranded, proteins, carbohydrates, viruses, cells, whole cells,organelles, organic polymers, other biological samples, particles,labeled media, labeled beads, and the like. The analytes can includebiomolecules, for example, cells, proteins, DNA, RNA, polynucleotides,polypeptides, polysaccharides, and small molecule analytes. According tovarious embodiments, the analyte can be a selected-sequencepolynucleotide, and an analyte-specific reagent including asequence-selective reagent for detecting the polynucleotide can beassociated with the polynucleotide. Polynucleotide analytes can bedetected by any suitable method, for example, polymerase chain reaction,ligase chain reaction, oligonucleotide ligation assay, hybridizationassay, antibody assay, affinity assay, or streptavidin/biotin assay.

FIG. 1 shows an exemplary embodiment of an electrophoresis device. Asdepicted in FIG. 1, the arrangement can include a channel-definingmember 10 defining a channel 12 therein for the migration of an analytesample. The channel-defining member 10 can include a cover plate with orwithout grooves, an etched plate defining one or more capillary sizedgrooves therein, or one or more capillary tubes. According to variousembodiments, the channel-defining member can be an etched plate having aplurality of channels or grooves, or the channel-defining member caninclude a plurality of capillary tubes. The use of a plurality ofchannels can allow a large number of analyte samples to be measuredsimultaneously in order to increase throughput. As is well known, forelectrophoresis to occur, opposing ends of channel-defining member 10,such as an electrophoretic plate or capillary tube, can be placed incontact with corresponding electrodes connected to a power supply forgenerating an electric field across the plate or tube. This field cancause the analyte to migrate from a loading site (not shown) for theplate or tube arrangement of the channel-defining member 10, toward adetection site or detection zone 14. The detection zone can encompassthat zone on the channel that is irradiated by an irradiation source toexcite markers, such as dye markers, used to label analytes in thesample.

An example of a marker compound can be a dye marker. Any suitablemarker, such as, for example, a fluorophore, can be used. Fluorophoresuseful according to various embodiments can include those that can becoupled to organic molecules, particularly proteins and nucleic acids,and that can emit a detectable amount of radiation or light signal inresponse to excitation by an available excitation source. Suitablemarkers can encompass materials having fluorescent, phosphorescent,and/or other electromagnetic radiation emissions. Irradiation of themarkers can cause them to emit light at varying frequencies depending onthe type of marker used.

One class of markers provides signals for the detection of labeledextension and amplification products by fluorescence, chemiluminescence,or electrochemical luminescence (Kricka, L. in Nonisotopic DNA ProbeTechniques, Academic Press, San Diego, pp. 3-28 (1992)).Chemiluminescent labels can include 1,2-dioxetane compounds (U.S. Pat.No. 4,931,223; and Bronstein, Anal. Biochemistry 219:169-81 (1994)).Fluorescent dyes useful for labeling probes, primers, and nucleotide5′-triphosphates include fluoresceins, rhodamines (U.S. Pat. Nos.5,366,860; 5,936,087; and 6,051,719), cyanines (Kubista, WO 97/45539),and metal porphyrin complexes (Stanton, WO 88/04777). Fluorescentreporter dyes include xanthene compounds such as fluoresceins I andrhodamines II:

The ring positions of I and II above can be substituted. The amino Rgroups of II can be substituted. The substituents can include covalentattachments to the primers, probes, or nucleotides. Examples of formulaeI and II include wherein X can be phenyl substituted with carboxyl,chloro, and other groups, for example, as described in U.S. Pat. Nos.5,847,162; 6,025,505; 5,674,442; 5,188,934; 5,885,778; 6,008,379;6,020,481; and 5,936,087, which are incorporated herein in theirentireties by reference, and wherein X can be hydrogen, for example, asdescribed in U.S. Pat. No. 6,051,719, which is incorporated herein inits entirety by reference.

According to various embodiments, an optical instrument can be providedthat includes a light source arranged to emit an excitation wavelengthor wavelength range toward a region capable of retaining a sample, suchthat a fluorescent dye, if present in the region, can be caused tofluoresce. The light source can provide excitation wavelength rangesthat correspond to respective excitation wavelength ranges of aplurality of fluorescent dyes. A detector capable of detecting anemission wavelength emitted from a fluorescing dye can be used todetermine the absence or presence of a component associated with thedye. For example, the dyes can include intercalating dyes, reporterdyes, free-floating dyes, and the like.

According to various embodiments, PCR dyes can be used that onlyfluoresce when bound to a target molecule. Nucleic acid sequenceamplification dyes can also be attached to probes that also areconnected to quenchers, and the action of nucleic acid sequenceamplification enzymes will disassemble the dye-probe-quencher moleculecausing the dye to increase its fluorescence. According to variousembodiments, nucleic acid sequence amplification can be performed usinga variety of methods, for example, polymerase chain reaction (PCR),isothermal amplification reaction, well known in the art. When a PCRprocedure is used, for example, the number of unquenched dye moleculesdoubles with every thermal cycle. Fluorescing dyes are well known in theart, and any of a plurality of fluorescent dyes having variousexcitation wavelengths can be used. Examples of such dyes include, butare not limited to, Rhodamine, Fluoroscein, dye derivatives ofRhodamine, dye derivatives of Fluoroscein, 5-FAM™, 6-carboxyfluorescein(6-FAM™), VIC™, hexachloro-fluorescein (HEX™), tetrachloro-fluorescein(TET™), ROX™, and TAMRA™. Dyes or other identifiers that can be usedinclude, but are not limited to, fluorophores and phosphorescent dyes.Dyes can be used in combinations of two, three, four, or more dyes persample. According to various embodiments, the family of 5-FAM™, 6-FAM™,VIC™, TET™, and/or ROX™ dyes can be used to indicate the presence ofsample components.

According to various embodiments, various detectable markers can beused, in addition or in alternate, to dyes. Markers can include, forexample, fluorescing dyes, free-floating dyes, reporter dyes, probedyes, intercalating dyes, and molecular beacons. Dyes that fluorescewhen integrated into DNA can be intercalating dyes. Other dyes known as“reporter” dyes can attached to the ends of “probes” that have“quenchers” on the other end. A nucleic acid sequence amplificationreaction, for example, PCR, can result in the disassembly of theDye-Probe-Quencher molecule, so the reporter dye can emit an increasedamount of fluorescence. Reporter dyes are not attached in any way to thesample. Free floating dyes can be floating freely in solution. Otherfluorescing markers well know in the art can be utilized. According tovarious embodiments, molecular beacons can be single-stranded moleculeswith hairpins that preferentially hybridize with an amplified target tounfold. According to various embodiments, quantum dots can be used asmarkers also.

In the embodiment shown in FIG. 1, an irradiation source is providedthat emits light in a given frequency range, such as, for example, alight emitting diode (LED) 16. It can be to be noted that, in theinstant description, the source of light can be any of a variety oflight sources. According to various embodiments, the light can have afrequency of about 660 nm or lower. The irradiation source can be, forexample, an LED, an organic LED, a non-coherent light source as known tothose skilled in the art, a solid state laser, a microwire laser, or acombination thereof. As used herein, the terms “irradiation source,”“light source,” “excitation source,” “LED,” or the like can includesingle or multiple sources of irradiation, including LED flood lightarrays. As used herein, “LED” can refer to an LED, an OLED, ormultiplicities thereof. Further, according to various embodiments, the“LED” can include coherent irradiation sources, for example, a solidstate laser source or a micro-wire laser source.

According to various embodiments, the light from the LED can bemodulated by an excitation modulating optics system before reaching thedetector zone 14. The excitation modulating optics system can include,as shown, a conditioning filter 18, the role of which can be tosubstantially block predetermined ranges of wavelengths of light emittedby the LED. The predetermined ranges can correspond to wavelengths oflight that can overlap with the emission spectra of the markers beingused. According to various embodiments, the conditioning filter can letthrough only light in the wavelength range of the excitation light ofone or more of the markers. Any given LED can emit excitation light in aspectral range. The range of wavelengths of the excitation light in turncan excite markers to emit light signals within a given spectral rangein the detection zone. For the detection of light signals from thedetection zone, that portion of the excitation light that would be inthe same wavelength range as some or all of the light signals emittedfrom the detection zone can be blocked. The light passing through theconditioning filter 18 is conditioned light 20, as seen in FIG. 1.

The excitation modulating optics system can further include a focusingoptical system 19. The conditioned light 20 can be focused by focusingoptical system 19 to irradiate the analyte sample and correspondingmarkers in the detection zone 14. The thus irradiated marker or markerscan turn emit light signals, such as through fluorescence, atfrequencies specific to the irradiated marker, so as to present a peakintensity. For example, a dye excited by yellow light can have afluorescence emission peak intensity at 610 nm corresponding to theorange portion of the spectrum. A peak intensity of about 460 nm can beassociated with the blue portion of the spectrum, and a peak intensityof about 660 nm can be associated with the red portion of the spectrum.By way of example, ROX, a known dye marker, can be best excited at 590nm. ROX can be excited by an LED emitting radiation at 590 nm.

The device of FIG. 1 can include a collection modulating optics systemthat can include a collimating optical system 24, a wide bandpass filter26, a transmission grating 28, and a re-imaging optical system 30.Emitted light 22 from the detection zone 14, and, in addition,conditioned light 20 passing through the detection zone 14, can becollimated by a first optical component or system 24. The light from thedetection zone can include the emitted light 22 and a portion of theconditioned light 20 passing through the detection zone. Alternatively,the excitation light can be brought in at an angle with respect to thedetection zone such that most of the conditioned light passing throughthe detection zone is not collected by the collimating optical system24. This reduces the excitation light that might be rejected. However,such an alternative arrangement also decreases the level of excitationlight that hits the detection zone 14. It can be, nevertheless, possibleto establish a compromise between irradiation angle and level ofexcitation light, as readily recognizable by those skilled in the art.

The light 20 and 22 from the detection zone 14 can be collimated bycollimating optical system 24. What is meant in the context of variousembodiments by collimation is any reduction in the propagation angle ofthe light being collimated. According to various embodiments, thereduction in the propagation angle of the light being collimated canresult in a propagation angle between about 20 degrees and about 0degrees.

According to various embodiments, a long pass filter, or, in thealternative, a wide bandpass filter 26, can be used for letting through,substantially exclusively, predetermined wavelengths of light from thedetection zone corresponding to a portion of wavelengths of the lightsignals emitted by an associated marker. The portion of the wavelengthsof the light signals can include, for example, all of the light signals,or it can include, for each marker, a range of wavelengths of the lightsignals, such as a range of wavelengths about the peak intensity of thelight signals. As an example, a wide bandpass filter can blockwavelengths of light outside of the range of from about 500 nm to about700 nm, thereby letting through only light that corresponds veryspecifically to the light emitted by the markers, that is, correspondingto emitted light 22. Thereafter, emitted light 22 can be spectrallydistributed by a transmission grating 28 and refocused by re-imagingoptical system 30 onto an array 34 of solid-state detectors, or detectorarray 34. The excitation modulating optics system and the collectionmodulating optics system are hereinafter collectively referred to as“the modulating optics system” or “modulating optics.” According tovarious embodiments, the array of solid-state detectors can include thephoto-detecting surface of the parallel register of a charge-coupleddevice (CCD) 32. As shown in FIG. 1, the image produced by refocusingemitted light 22 can be projected onto the detector array 34 of the CCD,producing a pattern of charge in proportion to the total integrated fluxincident on each pixel of the parallel register, as is well known in theart.

According to various embodiments, at least one of the excitation beampathway and the emission beam pathway can pass through a lens system,for example, a ball lens. The modulating optics system can include thelens system. For example, the excitation beam pathway and/or theemission beam pathway can pass through a lens system as described inU.S. patent application Ser. No. 10/805,096, filed Mar. 19, 2004, whichis incorporated herein in its entirety by reference. The modulatingoptics system can include or be separate from a lens system as describedin U.S. patent application Ser. No. 10/805,096.

Referring additionally now to FIGS. 2 and 3, an image produced by movinganalytes or analyte bands can be recorded by the photo-detecting surfaceof CCD 32 (FIG. 1) at times t (FIG. 2) and t+Δt (FIG. 3). Although theterm analyte band is used herein, it is to be understood that an analyteband can include a single analyte, such as a single cell. Thephoto-detecting surface 36 can be part of the two dimensional detectorarray 34 shown in FIG. 1. Photo-detecting surface 36 can include aspectral axis as indicated by arrow λ on the figure, and a spatial axisalong which the analyte bands move, as indicated by arrow M, in FIGS. 2and 3. As further seen in FIG. 2, an image created by the light signalsemitted by excited markers can produce, for example, two bands 38 and 40on photo-detecting surface 36, for example, substantially in the red andblue regions of the spectrum, respectively. Each band can correspond toa marker used to label, for example, a predetermined type of analyte.The bands can be spectrally distributed along the spectral axis bytransmission grating 28 as shown in FIG. 1. At time t, as shown in FIG.2, the charges produced on surface 36 can present two respective peaks46 and 48 on intensity profile 45. These peaks can correspond to bands38 and 40, respectively. As seen in FIG. 3, at time t+Δt, both bands 38and 40 can have moved downward along the direction of migration M on thephoto-detecting surface 36. Serial register 42 of the CCD 32 (FIG. 1)collects the charges accumulated for each analyte band during itsintegration time. All signals received from the detector can beconverted from analog to digital, and conveyed to a serial port fortransmission to a multipurpose computer for storage and/or for furtherprocessing and analysis. The analog output can alternatively oradditionally be sent directly to an output device for display orprinting, or used for other purposes.

According to various embodiments, for example, as shown in theembodiment above, collection of the image can be performed using timedelay integration (TDI). In the CCD, the photogenerated charge in thephotoactive elements or pixels can be transferred toward the serialregister 42 one row at a time. The charge information in the serial rowcan be read by using a corresponding single on-chip amplifier or readoutregister 44 of the CCD. By way of example, for a 256×256 element CCD,each time a single imaging area (one row) is transferred to the serialregister 42, 256 readouts of the thus transferred area are performed,each readout corresponding to a different spectral element or pixel inthe row. The above process continues until all 256 pixels in all 256rows have been read.

Under a normal read-out approach, the motion of the images on thedetector array 34 produces a blur. In TDI, according to variousembodiments, the shutter can be eliminated. The shifting of rows of theCCD can be synchronized to the migration of the band of analyte in thechannel. Thus, as an analyte band enters the excitation zone of the LED(detection zone 14), the light signals emitted therefrom can becollected and can illuminate the first row of the CCD, and thecorresponding charge information can be read using the amplifier orreadout register 44 of the CCD. The band takes a period of time, Atp, tomigrate in the channel so that its corresponding image migrates to thenext row of the CCD, one row closer to the serial register. After thistime period, the charge on the CCD can be shifted one row closer to theserial register, such that the fluorescence from the analyte correspondsto the same charge information on the CCD. Therefore, distinct from thephysical rows of the CCD, there exists in TDI according to variousembodiments a continuously moving row of accumulating photogeneratedcharge. An example of TDI in a capillary electrophoresis system usinglaser-induced fluorescence is disclosed in U.S. Pat. No. 5,141,609 toSweedler et al., and in J. F. Sweedler et al., Fluorescence Detection inCapillary Zone Electrophoresis Using a Charge-Coupled Device with TimeDelayed Integration, Anal. Chem. 63, 496-502 (1991), the contents ofboth of which are incorporated herein in their entireties by reference.

According to various embodiments, the effective integration time for agiven analyte band can vary from application to application. Theeffective integration time of a given analyte band can correspond to atime where the portion of the wavelengths of the light signals in theanalyte band being integrated moves across two pixels on the detectorarray, or to the entire time the portion of the wavelengths of the lightsignals in the analyte band being integrated is in the detection zone,or to any time therebetween. In addition, the portion of the wavelengthsof the light signals in the analyte band being integrated can, accordingto various embodiments, include (1) a range of wavelengths about a peakintensity of the light signals; (2) a range of wavelengths including allwavelengths of the light signals; or (3) a range of wavelengths anywherebetween (1) and (2) above. By way of example, the integration time caninclude a time it would take for the detector to integrate a range ofwavelengths of the analyte band corresponding to a full width of anintensity curve of the light signals in the analyte band at half of thepeak or maximum intensity of the intensity curve, or “full width at halfmax” of the intensity curve. The portion of the wavelengths of the lightsignals in the analyte band being integrated can depend on the number ofdifferent colors being integrated, and on how well the colors areseparated from one another in the emission spectra. As a general rule,the better separated the colors in the emission spectra, the wider theportion of the wavelengths of the light signals, and, hence, the longerthe effective integration time.

According to various embodiments, the use of TDI in collecting datapoints addresses the problem of lowered irradiance when usingirradiation sources emitting non-coherent light, such as LEDs. Theirradiance, that is, photons emitted per millimeters squared, can betypically about a thousand times lower in LEDs when compared with theirradiance of lasers. According to various embodiments, TDI addressesthe problem of lowered irradiance by allowing a longer period of timefor the integration of signals from excited markers. According tovarious embodiments, a broad detection zone can be used for TDI. In anon-TDI detection system, the detection zone can be typically about onetenth of a millimeter squared. When using TDI according to variousembodiments, the detection zone for one channel can be one hundred timeslarger, that is, about one millimeter squared, allowing a relativelylarger number of markers to be excited and a larger number of datapoints to be integrated into a detector. The various embodimentsdescribed herein can be equally applicable in instances where aplurality of channels are present, the detection zones of each of therespective channels being adapted to be irradiated by one or moreirradiation sources emitting light.

For the purpose of accumulating charges to effect TDI, instead ofshifting the charges on the CCD as a function of the migration of theanalyte bands, according to various embodiments, the CCD itself and/orthe image itself, that is, the light signals from the detection zone,can be moved as a function of migration of the analyte bands. The resultof such movement of the CCD and/or image can be the tracking of eachanalyte band by a continuously moving row of accumulating photogeneratedcharge on the CCD during the effective integration time of the analyteband. By way of example, to accomplish the desired result mentionedabove, appropriate motors, gearing, belt drives, control units and powersupplies can be used. For example, a linear actuator can be used totranslate the re-imaging optical system 30 and/or the CCD itself tominimize blurring. This can make the image stationary on the CCDthroughout the integration time.

According to various embodiments, the CCD can be a frame transfer CCD. Aframe transfer CCD has a parallel register that can include two CCDregisters arranged in tandem. The CCD register, or storage arrayadjacent to the serial register, can be covered with an opaque mask andcan provide temporary storage for charges during readout. The other CCDregister, or image array, identical in capacity to the storage array,can be used for imaging. After the image array is exposed to light, theresulting charges can be rapidly shifted in the parallel register up tothe storage array for subsequent readout. This shift operation typicallytakes a millisecond. While the masked storage array is being read, theimage array can integrate charge from the next image. See Charge-CoupledDevices for Quantitative Electronic Imaging, Photometrics Ltd. (1992),the content of which is incorporated herein in its entirety byreference.

To effect TDI, according to various embodiments, the modulating opticsas defined herein can be moved relative to a channel-defining member.For example, over a given detection zone, the modulating optics can bemoved along the detection zone at substantially the same speed as theanalyte for detection moves along the detection zone in thechannel-defining member. In this manner, the marker of a component ofthe analyte band can be repeatedly imaged on the same portion of thedetector, for example, a CCD, over the integration time. Alternately,the channel-defining member can be moved at the same speed but in anopposite direction from the movement of the analyte along thechannel-defining member. Movement of the channel-defining memberopposite the movement of the analyte to be detected can give theappearance of the analyte remaining stationary. Movement of thechannel-defining member opposite the direction of movement of theanalyte, in combination with movement of the modulating optics oppositethe direction of movement of the analyte to be detected, can be used incombination in order to accumulate data on all analytes in thechannel-defining member.

Referring now to FIGS. 4, 5 a, and 5 b, and according to variousembodiments, instead of one irradiation source, a plurality ofirradiation sources can be provided to excite marker compounds in asample. In the embodiment shown in FIGS. 4, 5 a, and 5 b, theirradiation sources can include four LEDs 50, 52, 54 and 56. The LEDscan be positioned so as to irradiate channel-defining member 58, whichcan define two channels in the form of, for example, two capillarytubes, as shown in FIG. 5 b. Each LED can emit non-coherent light in apredetermined range of wavelengths. For example, LED 50 and LED 52 canemit substantially blue light, LED 54 can emit substantially greenlight, and LED 56 can emit substantially yellow light. As the aboveexample shows, multiple LEDs can be used to increase the availablelight. For example, if LEDs 50 and 52 emit blue light, they can be usedsimultaneously, either continuously or in a pulsed fashion, in this wayincreasing the amount of available blue light to obtain a proportionalresponse in the associated markers. Although each type of marker usedcan ideally be excited by a different wavelength, LEDs of the optimumwavelength and power level for excitation of a given marker may not beavailable for each given application. Thus, different markers can beexcited by the same LED, according to various embodiments.

According to various embodiments, the modulating optics accordingdepicted in FIGS. 4, 5 a, and 5 b, can be comparable to the modulatingoptics in the embodiment shown in FIG. 1, with like components havingbeen labeled with like reference numerals. Thus, for each irradiationsource, a conditioning filter 18 and a focusing optical system 19 areprovided, it being understood that the respective conditioning filtersand focusing system for the respective irradiation sources are not,however, necessarily identical merely by virtue of the fact that theyhave been labeled with like reference numerals. As previously noted withrespect to FIG. 1, the function of each conditioning filter 18 can be tolet through only light in the wavelength range of excitation light forone or more of the markers. The conditioning filters 18 each cansubstantially block predetermined ranges of wavelengths of light emittedby the corresponding LED. The predetermined ranges correspond towavelengths of light that can overlap with the emission spectra of themarkers being excited by the corresponding LED. Each focusing opticalsystem 19 can focus the conditioned light from the conditioning filteronto the detection zone 14. As shown, for example, in FIGS. 4, 5 a, and5 b, the detection zone 14 can correspond to a respective detection zonefor each of the shown capillary tubes. Excited markers in detection zone14 can emit light signals in the form of emitted light 22. The lightfrom the detection zone can include the emitted light 22, and, inaddition, a portion of the conditioned light 20 passing through thedetection zone.

According to various embodiments, the light 20/22 from the detectionzone can be collimated by collimating optical system 24, as shown, forexample, in FIG. 5 a. The collimated light can be passed through acorresponding bandpass filter 51 on filter wheel 60 as shown in brokenlines in FIGS. 5 a and 5 b. It can be noted that the bandpass filters51, 53, 55, 57, and FD, in FIGS. 5 a and 5 b have been shown in brokenlines because, in those figures, the depiction of the filter wheel 60 isnot cross-sectional, but rather represents plan views thereof.

Referring to FIG. 4, the filter wheel 60 is shown in more detail, andincludes a plurality of bandpass filters 51, 53, 55, 57, and FD. Each ofthe bandpass filters can be adapted to let through, substantiallyexclusively, predetermined wavelengths of light from the detection zonecorresponding to a portion of the wavelengths of the light signalsemitted by an associated marker. According to various embodiments, therecan be a bandpass filter provided for each associated marker. Theportion of the wavelengths of the light signals allowed through arespective bandpass filter can include all of the light signals emittedby an associated marker, or it can include a range of wavelengths aboutthe peak intensity of the light signals emitted by the associatedmarker. For example, the range of wavelengths about the peak intensityof emitted light signals can be between about 5% and about 20% ofwavelengths on each side of the peak intensity for a given marker, or itcan include the range of wavelengths at about half of the peakintensity, full width at half max. For example, as shown in FIGS. 4, 5a, and 5 b, bandpass filter 51 can be adapted to filter therethroughlight signals emitted by given markers responsive to LED 50. In FIGS. 5a and 5 b, the apparatus can be depicted in a mode where LED 50 canirradiate the detection zone 14. However, any of the shown LEDs 50, 52,54, and 56, can be selectively used to irradiate the detection zone 14,or the detection zone 14 can be irradiated by more than one LED. Thefilter wheel 60 can be actuated by a filter wheel mechanism 62, as shownin FIG. 5 a. The filter wheel mechanism 62 can control the filter wheelto selectively position, in the path of the collimated light, thebandpass filter corresponding to the marker excited by the active LED,that is, by the LED being used to irradiate the detection zone. Filterwheel mechanism 62 can be controlled by a microprocessor or othersimilar device (not shown) as known to those skilled in the art. Thefiltered light can be focused by a re-imaging optical system 30 onto anarray 34 of solid-state detectors, or detector array 34, for example,the photo-detecting surface of the parallel register of a charge-coupleddevice, for example, CCD 32. According to various embodiments, a singleLED can be used to excite all markers, or multiple LEDs can be used withone LED exciting each respective marker, or multiple LEDs can be used toexcite a marker, or combinations thereof. The selection and number ofbandpass filters for use can be a function of the markers themselves,the number of LEDs, or other factors as knows to those skilled in theart.

According to various embodiments, and as shown in FIG. 4, the filterwheel can include a filter FD thereon, adapted to let through only lightsignals generated by a fifth marker. Four markers can be used to labelthe moving analytes where the analytes are DNA fragments, each markercorresponding to a given one of the bases in a DNA chain, that is,purines A (adenine) and G (guanine), and pyrimidines C (cytosine) and T(thymine). According to various embodiments, a fifth marker can, forexample, be used for fragment analysis of the analytes. The fifth markercan be any marker, such as a dye marker, for doing fragment analysis asknown to those skilled in the art. It is to be noted that, according tovarious embodiments, the number of markers that can be used are notlimited to four or five as stated in the above example, but are ratherlimited only by the number of dyes available on the market andresponsive to the irradiation source or sources being used based onapplication needs. Fragment analysis can be accomplished using, forexample, the GENESCAN® Analysis software produced by Applied Biosystems,Inc., Foster City, Calif. The GENESCAN® Analysis software calculates thesize of the unknown analytes by generating a calibration or sizing curvebased upon the migration times of the analytes in a standard that havebeen labeled with a marker. The unknown analytes can be mapped onto thecurve and converted from migration times to sizes. In the case of theembodiment shown in FIG. 4, the fifth marker filter FD on filter wheel60 can let through light signals corresponding to markers used to labelanalytes in the standard. These markers can be excitable by at least oneof the irradiation sources 50, 52, 54, and 56, appropriately mounted toallow fragment analysis.

FIGS. 6 a through 6 e depict images on the detector array 34 of CCD 32shown in the embodiment of FIGS. 4, 5 a, and 5 b, wherein the images canbe produced by moving analyte bands. Exemplary images are shown forexemplary markers that can be used to label the analytes, and that canbe responsive to excitation by a given one of the irradiation sources50, 52, 54, and 56. Each frame of photo-detecting surface 36 shown inFIGS. 6 a through 6e depicts two lanes of analyte bands, each lanecorresponding to one of the two capillaries of channel-defining member58. The bands move along the direction of migration M. The bands areshown in each of FIGS. 6 a through 6 e as being limited in the spectraldirection because the light recovered from the markers has been filteredthrough a corresponding bandpass filter 51, 53, 55, 57, or FD. At theright of each frame is an intensity profile 45 corresponding to chargeson the right lane of photo-detecting surface 36, which corresponds to acapillary of channel-defining member 58.

According to various embodiments, the intensity profiles can be alignedand combined according to known methods. The intensity profiles can bemulticomponented in order to account for any spectral overlap. Asdescribed with regard to the embodiment of FIG. 1, the serial registerof the CCD 32 in the embodiments depicted in FIGS. 4, 5 a, and 5 b,collects the charges accumulated for each analyte band during itsintegration time. All signals received from the detector can beconverted from analog to digital and conveyed to a serial port fortransmission to a multipurpose computer for storage, further processing,and analysis. Alternatively or additionally, the analog output can besent directly to an output device for display or printing. By way ofexample, a multipurpose computer can be used to perform themulticomponenting process. Multicomponenting is a process that is knownto those skilled in the art, and can involve a spectral calibrationwithin a multicomponenting software program. The spectral calibrationcan be obtained through a predetermined signature matrix correspondingto each marker. Each signature matrix can provide a signature snapshotof the intensity of light signals from a given marker as a function ofthe wavelengths of those light signals. By virtue of the signaturematrices, a combination of intensity curves for a given wavelength bandemitted from the detection zone can be broken down into its componentscorresponding to light signals emitted by individual ones of themarkers. In this way, a relatively accurate assessment of the lightsignals corresponding to respective ones of the markers can be made forthe detection process.

According to various embodiments, the apparatus as shown, for example,in FIGS. 4, 5 a, and 5 b, can irradiate detection zone 14 by irradiatingeach respective one of the irradiation sources 50, 52, 54, and 56, insequence. The filter wheel can be adjusted to dispose a bandpass filter51, 53, 55, 57, or FD, corresponding to the marker being used beforecollimating optical system 24. The detection zone can be irradiated forthe duration of the integration time, during which the analyte bandsmove across the detection zone. During the integration time, the chargesgenerated by the light signals from the markers to be detected can bemoved along a parallel register in the direction of migration. Thecharges can be accumulated in the detector or CCD 32 before they areread. Thereafter, the process can be repeated until all of theirradiation sources have irradiated the detection zone, and until allfilters, including filter FD, have been positioned before thecollimating lens to filter the light therefrom. Detector 32 can be aframe transfer CCD, wherein each frame of the CCD upon which chargeshave been accumulated can be transferred to a storage array, making theimage array available for the next series of charge accumulationsproduced by the next respective one of the irradiation sources beingused.

The above process can be repeated in cycles as many times as necessaryin order to obtain sufficient data regarding each analyte beingdetected. Fewer cycles can result in an increase in signal, becausefewer cycles mean longer integration times, and therefore increasedreadout signals over the noise typically associated with a CCD. On theother hand, increasing the number of cycles can improve the dynamicrange of the system. The dynamic range of the system is defined as thelargest peak signal that can be read by a given CCD (or “full wellcapacity”) over the smallest peak that can be read by the CCD just abovethe noise level. A CCD typically has a given full well capacity. If apeak signal is above the full well capacity of a CCD, it will be off thescale of the CCD. Short integration times allow peak signals to begenerally attenuated so as to reduce the possibility of saturating theCCD with off-scale signals, that is, with signals that go beyond theCCDs full well capacity. By using more cycles, analyte concentrationscan be increased while still allowing the CCD to reliably detect signallevels without saturation. According to various embodiments, there is atrade-off between using fewer cycles at a longer integration time suchas, for example, 5 seconds, and using more cycles at a shorterintegration time such as, for example, 1 second. Longer integrationtimes are useful where the noise level is relatively high and thesensitivity of the system needs to be increased because of the noiselevel. In systems where the noise level is relatively low, shorterintegration times allow multiple reads of signals from the same marker,and the read signals can be multicomponented, allowing the detection ofbrighter peaks without going off the scale of the CCD.

By way of example, a frame transfer CCD can collect light signalscorresponding to a blue marker during integration time t while an LEDexciting primarily the blue marker irradiates the detection zone.Thereafter, the entire CCD is read out. A filter wheel can be moved toposition a bandpass filter associated with a green marker before acollimating optical system, and an LED exciting primarily the greenmarker can irradiate the detection zone. The CCD can collect the lightsignals corresponding to the green marker during integration time t. Theentire CCD is then read out. The filter wheel can be moved to position abandpass filter associated with a yellow marker before a collimatingoptical system, and an LED exciting primarily the yellow marker canirradiate the detection zone. The CCD then collects the light signalscorresponding to the yellow marker during integration time t, and theentire CCD is thereafter read out. The above process can be repeated forall five markers, for example, as shown in FIGS. 4, 5 a, and 5 b. Theprocess can be repeated a number of times equal to the number ofmarkers. According to various embodiments, one or both of the LED andbandpass filter can be changed between CCD readouts. As suggested in theabove example, the image of the analyte band can take about five timesthe integration time to travel from the top of the frame transfer CCD tothe bottom thereof, that is, to the readout register. Each readout ofthe CCD can correspond to one marker. All of the readouts can be alignedand combined in a known manner for multicomponenting.

An example of multicomponenting is shown in FIGS. 6 f through 6 h. Forpurposes of FIGS. 6 f through 6 h, the filters 51, 53, 55, 57, and FD,are presumed to pass wavelengths of light in the blue, green, yellow,red and “fifth” portions of the spectrum, respectively. The “fifth”portion can, for example, be in the orange range of the spectrum. FIG. 6f is a schematic representation of an electropherogram showingfluorescence intensity curves during three readings of the signals bydetector 32, wherein each curve corresponds to light passed through arespective filter of the filter wheel in FIG. 4. The intensity curvescorrespond to a reading of light signals emitted from an analyte labeledwith a marker, for example, FAM, a dye marker that emits light signalsmostly in blue. The first portion of each curve, drawn in solid lines,corresponds to a reading from the respective blue filter, green filter,yellow filter, red filter, or fifth filter of FIG. 4 during a firstintegration time t. The second portion of each curve, drawn in brokenlines, corresponds to a reading from the respective filter during asecond integration time t. The third portion of each curve, drawn insolid lines, corresponds to a reading from the respective filter duringa third integration time t. In FIG. 6 f, the horizontal axis correspondsto distance traveled by the analyte, and the vertical axis corresponds,for each filter, to the fluorescence intensity of light emerging fromthat filter. Thus, the first set of curves in solid lines representsintensity curves for light passing through each filter during a firstcycle of the filter wheel 60. The second set of curves in broken linesrepresents intensity curves for light passing through each filter duringa second cycle of the filter wheel 60. The third set of curves in solidlines represents intensity curves for light passing through each filterduring a third cycle of the filter wheel 60. As seen in FIG. 6 f, thelight from the blue filter exhibits the most intensity during eachcycle, the intensity decreasing as light is collected from the greenfilter, the yellow filter, the red filter and the fifth filter,respectively. As seen in the instant example, therefore, spectraloverlap causes FAM to emit mostly in blue, some in green, less inyellow, etc. As filter wheel 60 is rotated within each cycle, to place asubsequent filter in the path of fluorescence from the detection zone14, the analyte moves a distance x as marked on FIG. 6 f.

FIG. 6 g is a schematic representation of the intensity curves of FIG. 6f in an aligned format for multicomponenting. Each intensity curve otherthan the one corresponding to blue light can be shifted by a multiple ofx, x being a function of the filter to be aligned with the intensitycurve corresponding to blue light. After being aligned, the intensitycurves are combined and multicomponented, yielding the intensity curvefor FAM shown in FIG. 6 h. To the extent that only FAM is being detectedin the example of FIGS. 6 f through 6 h, the intensity curves for markerdyes JOE, TAMRA, ROX, and the fifth dye, are shown as flat in FIG. 6 h.

FIGS. 6 i and 6 j demonstrate representative excitation efficiencycurves and fluorescence intensity curves, respectively, plotted versuswavelength for four different dye markers that can be used inoligosynthesis, namely, 5-FAM, JOE, TAMRA, and ROX. These dye markersare exemplary of those that can be used in various embodiments where aplurality of dye markers are to be used, such as with systems shown inFIGS. 4, 5 a, 5 b, and 7.

In FIG. 6 i, the x-axis corresponds to wavelengths, expressed innanometers (nm), emitted by an irradiation source. The y-axiscorresponds to the percentage of excitation efficiency. As shown in FIG.6 i, 5-FAM has a maximum absorbance, corresponding to its peak percentexcitation efficiency, at about 490 nm. When maximum absorbance of a dyemarker occurs at a given wavelength, it indicates that the dye markerfluoresces at its peak fluorescence intensity when it is irradiated atthat given wavelength. As further shown in FIG. 6 i, JOE has a maximumabsorbance at about 526 nm, TAMRA has a maximum absorbance at about 560nm, and ROX has a maximum absorbance at about 588 nm. FIG. 6 i alsoshows that, when a dye marker, such as 5-FAM, is irradiated at itsmaximum absorbance wavelength, other dye markers, such as, for example,JOE, TAMRA, and ROX, do exhibit some absorbance, although to a lesserextent than 5-FAM. Any irradiation source, for example, an LED, can beused to emit the wavelengths indicated on the x-axis.

In FIG. 6 j, the x-axis corresponds to wavelengths of fluorescent light,expressed in nm, emitted by excited dye markers. The y-axis correspondsto the percentage of fluorescence intensity. As seen in FIG. 6 j, 5-FAMhas a peak fluorescence intensity at about 522 nm, JOE has a peakfluorescence intensity at about 554 nm, TAMRA has a peak fluorescenceintensity at about 582 nm, and ROX has a peak fluorescence at about 608nm. The wavelengths on the x-axis are those that can be emitted by thefour mentioned dye markers. FIG. 6 j shows that where a dye marker, suchas TAMRA, fluoresces at its peak fluorescence intensity, other dyemakers, such as 5-FAM, JOE, and ROX, also fluoresce, although at lesserfluorescence intensities than TAMRA.

When different light wavelength sources are used, each dye marker in thedetection zone can be excited efficiently, and in a way that allowsdetection by its unique spectral signature. When two dye markers exhibitfluorescence intensity peaks that are close together, for example, whenthe difference between the fluorescence intensity peaks of the two dyemarkers is less than about 30 nm, there can be a high level of overlapof the light emitted by the two dye markers. A high level of overlap canmake it difficult to distinguish between the light emitted by the twodye markers, and therefore can make it difficult to determine therelative amounts of the two dye markers. Typically, there is overlappresent in the excitation and emission wavelengths of the different dyemarkers, as suggested, for example, in FIGS. 6 i and 6 j. However, theoverlap can be minimized by selecting dye markers that present easilydistinguishable fluorescence intensity peaks, such as those shown inFIG. 6 j. According to various embodiments, it is possible to firststart with an irradiation source emitting radiation within a given rangeof wavelengths, and to investigate each such irradiation source to seehow well it excites available dye markers. Graphs such as those shown inFIGS. 6 i and 6 j can be used in this context. For example, whereexcitation efficiency curves of various dye markers are plotted as shownin FIG. 6 i, an irradiation source, such as an LED emitting light in agiven range of wavelengths, can be predicted to excite given ones of thedye markers based on their excitation efficiencies. Using a fluorescenceintensity graph as shown in FIG. 6 j, the amount of overlap betweenintensity peaks of the different markers can be determined. Using thisinformation, a set of dye markers best suited for a particularapplication can be chosen. Once the dye makers are chosen, the filterscorresponding thereto can be chosen, for example, the filters shown onthe filter wheel of FIG. 4 described above, and on the filter wheel ofFIG. 7 described below. The best set of markers can exhibit the desiredminimum amount of overlap at the emitted wavelengths that are to bedetected.

Because each marker can have a different excitation curve, thefluorescence output of a marker can be dramatically increased by the useof an LED that is well matched to the excitation wavelength of themarker. Matching the light source and marker can increase emission fromthe marker of interest, while minimizing undesired light, for example,background light from the system and emissions from other markers,resulting in better quality data. For example, if a blue/green LEDhaving an emission wavelength of about 503 nm is used for the markerdesignated FAM, the excitation of FAM can be high, about 80%, and theexcitation for another marker, for example, ROX, can be low, forexample, about 6%. Similarly, a yellow LED with an emission wavelengthof about 592 nm will not excite FAM, but can excite ROX in an amount ofabout 90%.

Another embodiment of an apparatus is depicted in FIG. 7. As in theembodiments shown in FIGS. 4, 5 a, and 5 b, a number of irradiationsources can be used to sequentially irradiate a detection zone. Thegenerated light signals having wavelengths in differing frequency rangescan correspond to charges spatially offset on the same array of thedetector, wherein the offset is a function of the bandpass filter usedin connection with the markers emitting the light signals. Reading ofthe accumulated charges on the detector array during time delayintegration (TDI) can be continuous rather than a frame-by-framereading.

As shown in FIG. 7, the irradiation sources, together with associatedoptics such as the conditioning filter 18 and the optical system forfocusing 19, can be provided on an irradiation source wheel 61.Components of the apparatus of FIG. 7 that are similar to those in FIG.1 have been labeled with the same reference numerals, such asconditioning filter 18, focusing optical system 19, collimating opticalcomponent or system 24, and re-imaging optical component or system 30.An irradiation wheel 61 can be rotatable to selectively position eachrespective one of the irradiation sources and associated optics toirradiate a detection zone 14. Any number of irradiation sources, forexample, four irradiation sources 50, 52, 54, and 56, as shown, forexample, in FIGS. 4, 5 a, and 5 b, can be provided. A filter wheel 61′can be provided, similar to filter wheel 60 in FIGS. 4, 5 a, and 5 b.The rotation of both wheels 61 and 61′ can be effected by the provisionof a filter wheel drive 62 similar to the filter wheel drive of FIGS. 4,5 a, and 5 b, described above. According to various embodiments, the twowheels 61 and 61′ can be coupled to one another and to the filter wheeldrive 62 by way of a rotatable shaft 63. According to variousembodiments, a plurality of irradiation sources can be provided that arenot on an irradiation wheel. According to various embodiments, the twowheels 61 and 61′ can be actuated independently by their own respectivewheel drives, and are not coupled to one another by a shaft.

As shown in FIG. 7, the apparatus can be provided with an offset system64. Offset system 64 can either be disposed on the filter wheel 61′ inassociation with a corresponding bandpass filter, or can be coupled toat least one of a detector 32, one or more components of the modulatingoptics system, and the detection zone 14. The offset system 64 canspatially offset the light signals impinging upon the array 34 of thedetector 32 by a predetermined amount as a function of the bandpassfilter being used. The offset can be accomplished by an offset system 64including a plurality of offset mechanisms 66 disposed on filter wheel61′, wherein the offset mechanism 66 is shown in broken lines in FIG. 7.Each offset mechanism 66 can be associated with a respective one of thebandpass filters to offset the filtered light therefrom. The offsetmechanisms 66 can include one or more grating, mirror, prism, or anyother device for offsetting light as known to those skilled in the art,or a combination thereof. One or more offset mechanism 66 can bedistributed about the circumference of filter wheel 61′ adjacent thecorresponding bandpass filter, and between the bandpass filter and thedetector 32, otherwise being identical to wheel 60 in FIG. 4. In thealternative, the offset can be accomplished by providing an offsetsystem 64 including an offset control device 67, also shown in brokenlines in FIG. 7. The offset control device 67 can be coupled to at leastone of the detection zone 14, one or more component of the modulatingoptics system, and the detector array 34, in order to offset the lightsignals impinging upon the array 34 of the detector 32.

According to various embodiments, the offset system can move at leastone of the detection zone 14, collimating optical system 24, re-imagingoptical system 30, or detector array 34, by a predetermined amount inorder to offset the light signals impinging upon the detector array 34.The offset system can include any suitable device for effecting atranslational movement of at least one of the detection zone 14,collimating optical system 24, re-imaging optical system 30, or detectorarray 34, as known to those skilled in the art. Such devices caninclude, for example, solenoids, or motor driven linear actuators, forexample, lead screws, rack-and pinion systems, cams, and the like. Forexample, a cam can be attached to drive shaft 63 to cause apredetermined translation of the re-imaging optical system 30 in anumber of ways recognizable by those skilled in the art. Thepredetermined amount of translation can correspond in a one to one ratiowith the amount by which the light signals impinging upon the detectorarray are sought to be offset. The range of wavelengths offset by thepredetermined amount can be a function of the irradiation source, themarkers, or other factors known to those skilled in the art.

The predetermined amount by which a given set of light signalscorresponding to an irradiation source and its respective bandpassfilter is to be offset can be readily determined by determining where onthe detector array the charges produced by the given set of lightsignals corresponding to a specific bandpass filter should be situatedwith respect to the detector array itself, and with respect to sets oflight signals in different wavelength frequency ranges corresponding tothe other bandpass filters. Thus, where individual mechanisms 66 areused in conjunction with a corresponding bandpass filter to offset thelight emerging therefrom, each mechanism 66 can be chosen according tothe frequency range of wavelengths that the bandpass filter letsthrough. In the alternative, where offset control device 67 is used, theoffset control can be programmed to offset at least one of the detectionzone 14, one or more components of the modulating optics system, and thedetector array 34, with respect to one another by the predeterminedamount. The offsetting could, by way of example, be accomplished bymoving the detection zone 14, the modulating optics, or the detectorarray 34, in a translational motion by the predetermined amount, causingthe image created by the light signals to be correspondingly spatiallyoffset. The embodiment shown in FIG. 7 can involve the use of aplurality of LEDs similar to those used in the embodiment of FIGS. 4, 5a, and 5 b. The offset amount can be sufficient be prevent overlap ofthe images from each bandpass filter.

Offset system 64, including offset mechanisms 66, or, in thealternative, offset control device 67, is shown in broken lines in FIG.7 in order to suggest that mechanisms 66 or offset control device 67 canbe used as alternatives for the offset system 64. According to variousembodiments, both alternatives, that is, mechanisms 66 and offsetcontrol device 67, can be used in conjunction with one another. Themodulating optics can include at least one of conditioning filter 18,focusing optical system 19, collimating optical system 24, andre-imaging optical system 30, or any devices or systems known to achievethe functions associated with the components listed above as known tothose skilled in the art.

According to various embodiments, the detection zone can include anysuitable channel-defining member, for example, any number ofcapillaries, or any number of channels, in, for example, an etchedplate. The channel-defining member can be a slab plate. According tovarious embodiments, the channel-defining member can assume anyorientation according to application needs, for example, a horizontalorientation or a vertical orientation.

According to various embodiments, any suitable number of bandpassfilters can be used, the number being determined at least in part by themarkers being used. Any number of different irradiation sources, forexample, LEDs, that can emit light in any number of wavelength ranges,can be used. The number of LEDs can be dependent, in part, on the amountof available space, the availability of LEDs corresponding to theexcitation wavelength ranges of the marker(s), and/or the type ofmarker(s) that can be used.

As used herein, the term “optical system” can include a single lens, alens system, a mirror system, or any other optical system capable offulfilling the desired and stated functions, as readily recognizable bythose skilled in the art.

In operation, the detection zone 14 in FIG. 7 is irradiated by a firstone of the irradiation sources, for example, by LED 50, as depicted inFIG. 7. The light signals emitted by the markers excitable by the lightfrom LED 50 can be, as previously described, filtered through acorresponding bandpass filter 51, and thereafter focused onto detectorarray 34 by a re-imaging optical system. Where offset system 64 includesmechanism 66, the filtered light from bandpass filter 51 is offset by arespective mechanism 66 by the predetermined amount corresponding to therange of wavelengths that the bandpass filter lets through, as describedabove. Each subsequent irradiation source and corresponding bandpassfilter can be positioned to irradiate the detection zone and to filterthe light therefrom through a rotation of the wheels 61 and 61′ inconjunction with one another. As previously suggested, any suitablenumber of markers, bandpass filters, and LEDs can be used in the system.

In the embodiment of FIG. 7, charges can be accumulated for eachrespective irradiation source during the integration time of the analytebands excitable by light from the respective irradiation source. Theaccumulation of charges can be effected, as previously described, byshifting the charges on the detector array, by moving relative to oneanother the detector array and light signals from the detection zone, bymoving the modulating optics relative to the channel-defining member, ora combination thereof. After each integration time, the wheels 61 and61′ can be rotated to position the next irradiation source andcorresponding bandpass filter in a functional position, that is, in aposition for the irradiation source to irradiate the detection zone andfor the bandpass filter to filter the light from the detection zone. Theprocess can be repeated until one cycle is completed, that is, until allof the irradiation sources and filters have been used once. The cyclecan be repeated as many times as necessary and/or desired on anapplication-by-application basis. To the extent that the exemplaryembodiment of FIG. 7 includes an offset system for spatially offsettinglight signals in differing frequency ranges, the exemplary embodimentcan allow a continuous reading of accumulated charges by the detector,thereby making possible a continuous time delay integration of the lightsignals from the analytes into the detector array 34. Alternately, asshown in the exemplary embodiments of FIGS. 4, 5 a, and 5 b, theaccumulated charges corresponding to each range of wavelengths of lightsignals can be read and discarded by the detector before charges for thenext range of wavelengths are read.

FIG. 8 depicts an exemplary image that can be produced on the array ofdetector (CCD) 32 by the moving analyte bands in each of the twocapillaries 58 for each of the wavelength ranges. As depicted in theimage, each range of wavelengths is assigned an arbitrary color, forexample, “blue,” “green,” “yellow,” “red,” and “fifth.” For each color,the column on the left corresponds to charges produced by light signalsfrom one capillary, and the column on the right corresponds to chargesproduced by the second capillary. The array depicts charges generated bylight signals across the color axis λ, offset with respect to oneanother by offset system 64 in the manner previously described.According to various embodiments, the number of wavelength ranges usedcan be dependent on the particular application, and can range from oneto as many as the system supports. The λ axis is referred to here as the“color axis” rather than the “spectral axis,” because the colors, onefor each bandpass filter, do not need to be arranged from shorter tolonger wavelengths. According to various embodiments and as shown inFIG. 8, to the extent that charges from light signals of differingwavelengths can be produced on the same array of light signals so as tobe spatially offset with respect to one another, those charges can beaccumulated during the integration time and read by the detector on acontinuous basis. This can eliminate the need to shut off the detectorin order to allow a reading of charges in a given range of wavelengthson a frame-by-frame basis, and/or can eliminate the need for a frametransfer CCD. Further, a longer integration time and simpler data outputcan be achieved, and a cheaper CCD can be used.

According to various embodiments, the detection zone, including one ormore channels, or a slab gel, can be irradiated with multiple colorirradiation sources, for example, multiple color LEDs. The use ofmultiple color LEDs can greatly improve the absorption efficiency ofsome of the markers. According to various embodiments, an irradiationsource can be conditioned to provide only a narrow wavelength range oflight. A bandpass filter can be used to substantially block all of theunwanted excitation light from the irradiation source. As used herein,“irradiation source” can include one or more sources of irradiation. Anexample of the results, in the form of graphs demonstrating the effectsof a conditioned irradiation source in combination with a bandpassfilter, are illustrated in FIGS. 9 a through 9 d.

According to various embodiments and as depicted in FIGS. 9 a through 9d, a detection zone of an apparatus can be irradiated by two irradiationsources simultaneously, although any number of irradiation sources canbe used simultaneously according to various embodiments. A system suchas the one shown in FIG. 1 can be used, replacing the single LED in FIG.1 with a set of two or more LEDs. The associated optics can be alteredaccording to the number of irradiation sources used.

According to various embodiments, a conditioning filter can be used foreach set of LEDs. The conditioning filter can substantially blockpredetermined ranges of wavelengths of light emitted by the set of LEDsas previously described, each predetermined range corresponding to arespective LED, as demonstrated, for example, in FIG. 9 a. FIG. 9 ashows a graph of relative excitation intensity for each of two LEDs of aset of LEDs, versus wavelength expressed in nanometers. The graph ofFIG. 9 a depicts the two LEDs as being in the violet and orange portionsof the spectrum, respectively. It is to be understood that variousembodiments are not limited to the above two types of LEDs, butencompasses any number of LEDs emitting light in any range offrequencies according to application needs. As depicted in FIG. 9 b,each predetermined range of wavelengths that passes through theconditioning filter corresponds to a subset of the wavelengths emittedby each respective LED depicted in the graph of FIG. 9 a. Thewavelengths that pass through the conditioning filter can be capable ofexciting the markers responsive to each respective LED. FIG. 9 b is agraph of percent transmission of light through the conditioning filterversus wavelength. As shown in FIG. 9 b, the conditioning filter cansubstantially block all excitation light except for wavelengths aroundthe respective LED emission maximum. For example, the conditioned lightranges can correspond to a wavelength range of from about 450 nm toabout 490 nm, and to a second wavelength range of from about 580 nm toabout 605 nm, corresponding to the first and second irradiation sources,respectively. The conditioning filter, and the predetermined rangescapable of passing therethrough, can be functions of the LED set beingused.

According to various embodiments, the conditioning filter can include asingle conditioning filter, or a series of conditioning filters, capableof filtering the light from the set of LEDs. The conditioned light canbe focused onto the detection zone of an electrophoretic detectionsystem, as previously described above. The light emitted by markers inthe detection zone can be passed through a bandpass filter. The bandpassfilter can allow, substantially exclusively, predetermined wavelengthsof light from the detection zone to pass through, wherein thepredetermined wavelengths of light correspond to a portion of thewavelengths of the light signals emitted by an associated set ofmarkers. The light that passes through the bandpass filter can befiltered light. The bandpass filter can substantially block theexcitation light from the LEDs which passes the detection zone. Theportion of the wavelengths of the light signals that can pass throughthe bandpass filter can include all of the light signals, or a range ofwavelengths about the peak intensity of light signals of each respectivemarker. For example, the range of wavelengths about the peak intensityof light signals can be between about 5% and about 20% of wavelengths oneach side of the peak wavelength of a given marker, or it can includefull width at half max. The collection of the predetermined wavelengthscan be by dispersion, for example, as shown in FIG. 1, or by use ofadditional bandpass filters, as shown, for example, in FIG. 4.

According to various embodiments, a conditioning filter can be usedbetween the irradiation source (light source) and the detection zoneholding a sample solution. The beam emitted by the light source is alsoknown as an excitation beam that can be filtered by an excitation filterat an excitation wavelength range. Comparably, a conditioning filter canbe used between the detection zone holding the sample solution and adetector. The fluorescence or luminance emitted by the sample uponexcitation is also known as an emission beam that can be filtered by anemission filter at an emission wavelength range.

According to various embodiments, a light source can be used to provideexcitation beams to irradiate a sample solution containing one or moredyes. For example, two or more excitation beams having the same ordifferent wavelength emissions can be used such that each excitationbeam excites a different respective dye in the sample. The excitationbeam can be aimed from the light source directly at the sample, througha wall of a sample container containing the sample, or can be conveyedby various optical systems to the sample. An optical system can includeone or more of, for example, a mirror, a beam splitter, a fiber optic, alight guide, or combinations thereof.

According to various embodiments, one or more filters, for example, abandpass filter, can be used with a light source to control thewavelength of an excitation beam. One or more filters can be used tocontrol the wavelength of an emission beam emitted from an excited orother luminescent marker. One or more excitation filters can beassociated with a light source to form the excitation beam. One or morefilters can be located between the one or more light sources and asample. One or more emission filters can be associated with an emissionbeam from an excited dye. One or more filters can be located between thesample and one or more emission beam detectors.

According to various embodiments, one or more filters, for example, abandpass filter, can be used with a light source to control thewavelength of an excitation beam. One or more filters can be used tocontrol the wavelength of an emission beam emitted from an excited orother luminescent marker. One or more excitation filters can beassociated with one or more light sources to form at least oneexcitation beam. One or more filters can be located between the one ormore light sources and a sample. According to various embodiments, oneor more emission filters can be associated with an emission beam from anexcited dye. One or more filters can be located between the sample andone or more emission beam detectors.

According to various embodiments, a filter can be used that is a singlebandpass filter or a multiple bandpass filter. As used herein, abandpass filter and a passband filter are used interchangeably. Amultiple passband filter can be, for example, a multiple-notch filter ora multi-Rugate filter. A multiple passband filter can be used with anincoherent light source, for example, a halogen lamp, a white lightsource, and/or one or more LEDs or OLEDs emitting light at differentwavelengths. A multiple passband filter can be used with a multiplelaser-based light source emitting light at different wavelengths.Examples of manufacturing and use of Rugate filters and Rugate beamsplitters can be found in, for example, U.S. Pat. No. 6,256,148 toGasworth which is incorporated herein by reference in its entirety.

According to various embodiments, a multiple passband filter can be usedwith a dichroic beam splitter, a 50/50 beam splitter, a dichroic beamsplitter that has several “passbands,” or no beam splitter. A multiplebeam splitter can be coated at an angle, causing a variance in athickness across a filter substrate, to compensate for wavelength shiftwith an angle. A beam splitter can be disposed at a low angle along alight or beam path to sharpen edges of the filtered light and can easefabrication. A beam splitter can be disposed at a 45° angle of incidencealong a light beam path. A low angle can include an angle of incidenceless than 45°, less than 30°, or less than 15°. A multiple passbandfilter can be formed by coating different light interference materialsover respective areas of a substrate used in a multiple passband filtermanufacture.

A Rugate filter is an example of an interference coating based on therefractive index that varies continuously in a direction, for example,perpendicular or 45 degrees to the film plane. When the refractive indexvaries periodically within two extreme values, a minus filter with hightransmittance on either side of the rejection band can be made. PeriodicRugate filters can be manufactured.

Rugate notch filters can use refractory metal oxides to achieve coatingswith exceptional thermal and environmental stability. These filters canbe used in place of other types of notch filters, particularly wheredurability and reliability are desired. Rugate notch filters areavailable from Barr Associates (Westford, Mass.). The Rugate notchfilter can be used as edge filters and beam splitters. Filter sizes orshapes are not limitations for the Rugate notch filter. The Rugate notchfilter can provide environmental and thermal stability, a broadoperating temperature range, narrow rejection bands, variety of shapes &sizes, high throughput, low ripple, and/or a broad spectral range. Moreinformation is available from, for example, www.barr-associates-uk.com,www.barrassociates.com/opticalfilters.php.

Multiple-notch filters can be made, for example, with a measuredblocking of O.D. 6 or better. Notch filters with this type of deepblocking level at the light wavelength can also afford high transmissionclose to the light line.

According to various embodiments, excitation levels can increase whenmultiple dyes spaced apart spectrally are irradiated with excitationbeams. This can lead to less spectral crosstalk. The dye matrix,condition number, and/or deconvolution in a system can be improved. Theincreased excitation levels can provide higher signal levels. Highersignal levels can be seen during the utilization of dyes that emit inthe “red” spectrum. The dynamic range of the system can be improved. Thesystem can reduce the compensation for variation in the emission beamintensity for various dyes.

FIG. 9 c depicts percent light transmission plotted versus wavelength.According to various embodiments, the bandpass filter can allow lightcorresponding to the “blue,” “green,” “yellow,” “red,” and “orange”markers to pass through. The regions or zones corresponding to theexcitation light of the LEDs can be blocked. According to variousembodiments, the bandpass filter can include a single bandpass filter, aseries of bandpass filters capable of filtering the light from the LEDs,a multi-notch filter, or a Rugate filter.

FIG. 9 d depicts a plot of relative emission intensity versuswavelength, expressed in nm, for the filtered light. FIG. 9 d, ineffect, provides a breakdown, by wavelength, of the light transmittedthrough the exemplary bandpass filter. As shown in FIG. 9 d, the markersexcited by the LEDs used in the example of FIGS. 9 a through 9 d emitlight in the “blue,” “green,” “yellow,” “red,” and “orange” ranges ofwavelengths. Before focusing the light signals thus filtered onto thedetector array of a CCD, a dispersion element can be used, such asgrating 28 shown in FIG. 1. The resulting image on the CCD can besimilar to that depicted in FIGS. 2 and 3, with the addition of one ormore dark zones corresponding to the blocked excitation lightwavelengths. The existence of one or more dark zones, however, does notprevent the performance of multicomponenting to determine the intrinsicdye concentrations of each band in an electropherogram. TDI can beperformed during data collection with the above-described systemembodiments.

According to various embodiments, when irradiating multiple-channelswith multiple color LEDs, the channels can be selectively masked inorder to keep the light signals generated by the markers in each channelseparate from one another. For example and as shown in FIGS. 10 and 11,in an embodiment where three channels 68 a, 68 b, 68 c, are provided,each channel can be selectively masked with a mask 70 to create threewindows 72 a, 72 b, and 72 c, in the irradiation zone 67. The mask canbe made of a metallized surface on a glass or fused silica plate that isdisposed adjacent, for example, immediately above, the channels. Themetal can be deposited in a controlled manner, for example, byphotolithography methods, to cover the entire plate except in the areasforming the windows 72 a, 72 b, and 72 c. Alternatively, windows can becut out of an optically non-transparent plate, or a plate with windowscan otherwise be formed as known to those skilled in the art.

As depicted in FIG. 11, the array 34 of a detector can be configuredsuch that it is divided into three frames 74 a, 74 b, and 74 c,respectively, corresponding to windows 72 a, 72 b, and 72 c, allowingTDI to be effected on the charges generated by light signals detectedthrough each respective window on a window-by-window basis. The numberof wavelengths can correspond to the number of channels, or can be lessthan the number of channels. The mask 70 can be a single mask withmultiple windows, or a series of masks, each mask corresponding to oneor more channels. A device with any number of channels can be masked.According to various embodiments, each channel can pass through one ormore excitation zones. Each excitation zone can be read by acorresponding detector or emissions from each exciation zone can passthrough a corresponding detection zone.

FIG. 12 a is a schematic cut-away representation of a portion ofdetector array 34 corresponding to frame 74 a for detecting lightpassing through window 72 a. As shown in FIG. 12 a, each charge 76corresponding to an analyte band moves in the migration direction M, andthe charge packets on the detector array 34 are accumulated over anintegration time. Charges can be accumulated for TDI by shifting thecharges on the detector array in the migration direction M at apredetermined speed corresponding to the average speed of migration ofthe analyte bands, as suggested in FIG. 12 a by the shifting movementsdepicted by reference numeral 78. Data can be collected from frame 74 afrom the top of the frame to the bottom of the frame. Some of the lightcan be blocked by the mask, causing a variable collection efficiency, asdepicted by FIG. 12 b. The integration of the accumulated charges canoccur on a frame-by-frame basis as previously explained in relation toFIGS. 4, 5 a, and 5 b, the frame data being combined in a known mannerto create an electropherogram. It is further possible, according tovarious embodiments, to abut the frames, that is, to eliminate anydistance between them so as to combine the resulting images on detectorarray 34. According to various embodiments, the above-describedarrangements can allow the separation and detection of light signalsfrom multiple channels while permitting the simultaneous irradiation ofthose channels with multiple color irradiation sources. According tovarious embodiments, a single camera can be used to maintain a spatialseparation of light signals from each masked channel, or one camera perchannel can be used.

According to various embodiments, modulating optics useful for theelectrophoresis arrangements described herein are disclosed in U.S.application Ser. No. 09/564,790, the contents of which are incorporatedherein in their entirety by reference. For example, the modulatingoptics shown in FIG. 1 of U.S. patent application Ser. No. 09/564,790,using a cat's eye aperture of FIG. 24, can be useful in variouselectrophoresis arrangements as described herein.

According to various embodiments, an apparatus for detecting analytes ina sample can include means defining one or more channels therein havinga detection zone; means for separating a sample into analytes migratingalong the one or more channels, wherein the sample can include analytesand can be disposed in contact with a migration medium disposed withinthe one or more channels, wherein each analyte is detectable by thepresence of a marker; means for irradiating the detection zone withnon-coherent radiation, that can thereby excite markers responsive tothe radiation and which markers can emit light signals indicative ofcorresponding analytes; means for detecting the light signals bycollecting the light signals and producing corresponding charges; meansfor effecting a time delay integration of the charges produced by thelight signals within a detector array by accumulating within thedetector array the charges corresponding to light signals associatedwith at least one given analyte during an integration time of the atleast one given analyte moving across the detection zone; means forreading the accumulated charges; and means for separating the analytesbased on the accumulated charges that are read. Various embodiments ofthe above-described means have been substantially shown in and describedwith relation to FIGS. 1 through 12 b.

According to various embodiments, the apparatus can include a sortingmechanism to affect flow cytometry, or fluorescence-activated cellsorting, of the detected components of the sample in thechannel-defining member. The components can be cells, blood cells,nucleic acid sequences, or other biological sample components. Thesorting mechanism can be located after the detection zone. The sortingmechanism can be located a suitable distance from the detection zone toallow sufficient time for determination of the composition of acomponent of the sample based on the detected charges receivedcorresponding to the light signals associated with the at least onecomponent of the sample. Based on the identification of the component,the component can be directed into one of two or more channels. Allcomponents of a certain composition, charge, or other identifyingfactor, can be directed into the same respective channel.

The sorting mechanism can include, for example, use of an electrostaticforce, mechanical movement of respective channels, use of an acousticpulse to divert the detected component to a respective channel,manipulation of electric field gradients, vacuum, or other means asknown to those of ordinary skill in the art. Various examples of suchmethods of sorting are shown in FIGS. 13 a-c. For example, FIG. 13 adepicts a channel 12 including a detection zone 14 through whichradiation 20 passes. Emission radiation signals 22 can emit along thesame path as traveled by radiation 20, or along a separate path (notshown). Emission radiation signals 22 traveling along the same path asradiation 20 can be separated by using modulating optics as describedherein. Emission radiation signals 22 can be detected, for example, bydetection array 34 of FIG. 1 and FIG. 5 a. An analyte or component 80can travel via the channel 12 past the detection zone 14. A diverter,for example, a charge inducer 85 can be situated at a distance past thedetection zone, sufficient for the detector to determine the compositionor salient features of the component 80. The charge inducer 85 can applya no charge, a negative charge, or a positive charge to the component oranalyte 80. The component or analyte 80 having received a charge or nocharge from the charge inducer 85 can pass out of channel 12 and traveltowards one or more containers, for example, depicted as LEFT, WASTE,RIGHT in FIG. 13 a. The one or more containers can correspond, forexample, to a position directly below channel 12 for receipt of thenon-charged components, to the left of the channel 12 for receipt of acharged component 80, and to the right of the channel 12 for receipt ofan oppositely charged component 80. To direct the charged components 80,a magnetic field or electric field gradient can be applied across thearea through which the components fall towards the containers.Negatively charged components can gravitate towards the positivelycharged side of the field, or a positively charged plate, and positivelycharged components can gravitate towards the negative side of the fieldor the negatively charged plate, being directed into their respectivecontainers.

FIG. 13 b depicts an alternate embodiment of a sorting mechanism. Ananalyte or component 80 can travel through a channel 12 having adetection zone 14 through which radiation 20 passes, emitting emittedlight 22. Radiation 20 traveling along a direction before the detectionzone 14 can continue traveling along the same direction after thedetection zone 14 towards a detector (not shown). The detector and thedetector's associated radiation modulating optics can be disposedtransverse from the direction of the radiation 20, rather than along it.This transverse placement of the detector can improve detection ofemitted light 22. Emitted light 22 can be detected in the presence ofradiation 20. Emitted light 22 can be detected after separation fromradiation 20. After passing through the detection zone 14, the component80 can travel some distance before leaving the channel 12. The distancetraveled can be related to the time necessary to determine thecomposition or salient features of the component having passed throughthe detection zone 14. The composition of the component can cause adiverter 83 to divert the component 80 to a branch channel 88 orcontinue flowing along the channel 12. The diverter 80 can allowdetected component 80 to flow along channel 12. According to variousembodiments, one or more mechanical arms having a collection tube, acollecting container, or other means of collecting the component, can beused to move into position below channel 12 to collect the designatedcomponent 80. A device to move a collection tube, for example, a seriesof mechanical arms, can be used to collect components of variousnatures, in different collection tubes. A waste container or collectingbin can be located below channel 12 to collect any components not placedin a container of one of the mechanical arms. According to variousembodiments, the component can be removed from the stream of componentsleaving channel 12 into a desired branch channel 88 by some force, forexample, an acoustic force, a vacuum force, a pneumatic force, or someother means of moving the particle out of the particle stream and into adesignated container. According to various embodiments, a switchableelectric field or varying current flow can be applied to or across thebranch channel 88 using multiple electrodes, for example. The desiredbranch channel 88 can have a voltage potential across it, the otherbranch channels being allowed to “float” electrically. When a potentialis present at the desired branch channel the analyte can flow into theintended branch channel. A movement of the detected component 80 intothe electrically floating branch channels can be prevented. An analytepreviously moved to a branch channel 88 can be prevented from movinginto channel 12.

FIG. 13 c is another embodiment of a sorting system. The particles 80can travel in a channel 12 and pass through a detection zone 14. Channel12 can be in communication with branch channels or side channels 82, 84,and 86, branching therefrom. After passing the detection zone 14, acomponent 80 can travel down channel 12 for a distance sufficient foranalysis and recognition of the component 80 by the sorting system. Thecomposition of the component 80 can be used to determine a side channel82, 84, 86 for the component 80 to traverse. The component 80 can remainin the channel 12. Diverters 83′, 83″, and 83′″ can be activated todivert component 80 into one of branch channels 82, 84, 86. Thecomponent 80 can be maneuvered into the correct or corresponding branchchannel 82, 84, 86, or the component 80 can be allowed to continue alongchannel 12, by manipulation by electric field gradients, switchableelectric fields, vacuum forces, a stream or air, or by other means ofmovement as known to those of ordinary skill in the art. In particular,the device illustrated in FIG. 13 c can be part of a microdevice (notshown), for example, a microcard (not shown) containing channels 12, 82,84, and 86 as microcapillaries.

According to various embodiments, a sorting mechanism can be activatedupon detection of a component or analyte. The activation can cause adetection component to be removed from a flow stream containing thecomponent. After a delay time, a diverter can direct the detectioncomponent to be removed from the flow stream into a container or acollection bin. According to various embodiments, any sample notdirected to a container can be discarded as waste or can be used forsample recovery. According to various embodiments, one or more kinds ofcomponents can be present in the flow stream. One or more kinds ofcomponents can be detected. The diverter can direct variouslyidentified/detection components into one or more containers. Thediverter can employ a variety of methods to sort and/or collectcomponents. The methods of sorting the components, also known asconducting flow cytometry or cytofluorometry, can include the use ofmotive forces, for example, electrostatic forces, mechanical movement,electric field gradients, swiitchable electric fields, vacuums, streamsof air, or other motive forces as known to those of ordinary skill inthe art. According to various embodiments, the containers collecting thecomponents can be wells in a microdevice, for well, a microcard inmicrotiter format including one or more, for example, 24, 48, 96, 192,384, or more, wells. A sorter suitable for component sorting, forexample, cells, nucleotide acid sequences, can be obtained formDakoCytomation of Fort Collins, CO under the registered name MOFLOSorters. Further background on flow cytometry and cell sorting can beobtained in, for example, Hoy, Cell Sorting. Principles,http://www.uwcm.ac.uk/study/medicine/haema-tology/cytonetuk/documents/sort.pps(printed Apr. 14, 2004), which is incorporated herein its entirety byreference.

As shown with respect to FIGS. 13 a-c, multiple methods of manipulatinga component 80 for sorting the component based on its composition orbased on other features, can be used after component 80 has passeddetection zone 14. Flow cytometry can include coordination of thedetector, the processor for analyzing the data received from thedetector, and a control unit for controlling the motive force used tomanipulate the movement of the component 80. The flow cytometer caninclude a control for receiving and acting on a control signal that issent from a corresponding detector or time-delay integration system.Further description of optical flow cytometers can be found, forexample, in U.S. Pat. No. 6,549,275 which is incorporated herein in itsentirety by reference.

It is to be understood that various embodiments can be useful indetecting and imaging not only fluorescent labeled molecules, but alsoother chemical and/or biological cells or molecules, for example,proteins, viruses, and bacteria, nucleic acid sequences. Variousembodiments can be useful for the detecting and imaging of componentswhich can be electrophoretically or otherwise separated on a variety ofcarriers, for example, in capillary tubes, and across, on, in, orthrough slab gels, membranes, filter paper, Petri dishes, glasssubstrates, and the like.

According to various embodiments, the light source can be a LightEmitting Diode (LED). The LED can be, for example, an Organic LightEmitting Diode (OLED) an inorganic Light Emitted Diode, that can bepolymer-based or small-molecule-based (organic or inorganic), an edgeemitting diode (ELED), a Thin Film Electroluminescent Device (TFELD), ora Quantum dot based inorganic “organic LED.” The LED can include aphosphorescent OLED (PHOLED). As used herein, the terms “excitationsource,” “irradiation source,” and “light source” are usedinterchangeably.

According to various embodiments, excitation beams emitted from thelight source can diverge from the light source at an angle ofdivergence. The angle of divergence can be, for example, from about 5°to about 75° or more. The angle of divergence can be substantially wide,for example, greater than 45°, yet can be efficiently focused by use ofa lens, such as a focusing lens.

According to various embodiments, the light source can include one LightEmitting Diode (LED) or an array of LEDs. According to variousembodiments, each LED can be a high power LED that can emit greater thanor equal to about 1 mW of excitation energy. In various embodiments, ahigh power LED can emit at least about 5 mW of excitation energy. Invarious embodiments, the LED or array of LEDs can emit, for example, atleast about 50 mW, at least about 500 mW, or at least about 1 W or moreof excitation energy. A cooling device such as, but not limited to, aheat sink or fan, can be used with the LED. An array of high-poweredLEDs can be used that draws, for example, upto about 10 watts of energyor more, upto about 100 watts of energy or more, or up to about 1000watts of energy or more. The total power draw can depend on the power ofeach LED and the number of LEDs in the array. The use of an LED arraycan result in a significant reduction in power requirement over otherlight sources, such as, for example, a 75 watt halogen light source or a150 watt halogen light source. Exemplary LED array sources areavailable, for example, from Stocker Yale of Salem, N.H. under the tradename LED AREALIGHTS, or from Lumileds Lighting, LLC of San Jose, Calif.under the trade name LUXEON, for example, a LUXEON STAR. Examples ofLEDs can be found athttp://www.lumileds.com/products/family.cfm?familyld=1. According tovarious embodiments, LED light sources can use about 1 milliwatt (mW) ormore of power, for example, about 25 mW or more, about 50 mW or more,about 1 W or more, about 5 W or more, about 50 W or more, or about 100 Wor more, individually or when in used in an array.

According to various embodiments, a quantum dot can be used as a sourcefor luminescence and as a fluorescent marker. Quantum dots can be usedfor both. The quantum dot based LED can be tuned to emit light in atighter emission bandpass, for example, an emission bandpass including afull-width of half-max of about 10 nm or less, about 20 nm or less, orabout 50 nm or less. The quantum dot based LED can increase theefficiency of the fluorescent system. The efficiency of a quantum dotbased LED can theoretically be higher than that of conventional LEDs,potentially over 90% when sandwiched directly between two conductivefilms with each film directly touching each quantum dot as opposed tothe present 20% efficiency for standard LEDs. Quantum dot based LEDs canbe made utilizing a slurry of quantum dots, where current flows throughan average of several quantum dots before being emitted as a photon.This conduction through several quantum dots can cause resistive lossesin efficiency. Quantum dots can provide many more colors thanconventional LEDs.

Quantum dots can be molecular-scale optical beacons. The quantum dotnanocrystals can behave like molecular LEDs (light emitting diodes) by“lighting up” biological binding events with a broad palette of appliedcolors. Quantum dots can provide many more colors than conventionalfluorophores. Quantum dots can possess many other very desirable opticalproperties. Nanocrystal quantum dots can be covalently linked tobiomolecules using standard conjugation chemistry. The quantum dotconjugate can then be used to detect a binding partner in a wide rangeof assays. According to various embodiments, streptavidin can beattached to quantum dots to detect biotinylated molecules in a varietyof assays. Quantum dots can also be attached to antibodies andoligonucleotides. Any assay that currently uses, for example,fluorescent-tagged molecules, colorimetric enzymes, or colloidal gold,can be improved with quantum dot nanocrystal-tagged conjugates. Anexemplary quantum dot implementation is available from Quantum DotCorporation of Haywood, Calif. under the trademark QDOT. Moreinformation about quantum dots and their applications can be found at,for example, www.qdots.com, and in U.S. Pat. Nos. 6,207,229, 6,251,303,6,306,310, 6,319,426, 6,322,901, 6,326,144, 6,426,513, and 6,444,143 toBawendi et al., U.S. Pat. Nos. 5,990,479, 6,207,392, and 6,423,551 toWeiss et al., U.S. Pat. No. 6,468,808 to Nie et al., and U.S. Pat. No.6,274,323 to Bruchez et al., which describe a variety of biologicalapplications, methods of quantum dot manufacturing, and apparatuses forquantum dot nanocrystals and conjugates, all of which are incorporatedherein by reference in their entireties.

Quantum dots can provide a versatile probe that can be used in, forexample, in multiplex assays. Fluorescent techniques using quantum dotnanocrystals can be much faster than conventional enzymatic andchemiluminescent techniques, can reduce instrument tie-up, and canimprove assay throughput. Colorimetric or detected reflectancetechniques can be inferior to fluorescence and difficulties ensue whenmultiplex assays are developed based on these materials. Quantum dotscan absorb all wavelengths “bluer” (i.e., shorter) than the emissionwavelength. This capability can simplify the instrumentation requiredfor multiplexed assays, since all different label colors can be excitedwith a single excitation source.

A Quantum dot based LED can emit light in an emission band that isnarrower than an emission band of a normal LED, for example, about 50%narrower or about 25% narrower. The emission band of the quantum dotscan be a function of the size distribution of the quantum dots, and thuscan theoretically be extremely narrow. The Quantum dot based LED canalso emit light at an electrical energy conversion efficiency of about,90% or more, for example, approaching 100%. OLED films, includingQuantum dot based LEDs, can be applied to a thermal block, used forheating and cooling samples, in a fluorescence system withoutinterfering with the operation of the thermal block.

According to various embodiments, when an OLED is used, the OLED canhave any of a variety of sizes, shapes, wavelengths, or combinationsthereof. The OLED can provide luminescence over a large area, forexample, to luminescence multiple sample wells. Scatter or cross-talklight between multiple sample wells for this single OLED can be reducedby either overlaying a mask on the OLED or by patterning the luminescentin the OLED to operatively align with the multiple sample wells. TheOLED can be a low power consumption device. Examples of OLEDs in variousconfigurations and wavelengths are described in, for example, U.S. Pat.No. 6,331,438 B1, which is incorporated herein by reference in itsentirety. The OLED can include a small-molecule OLED and/or apolymer-based OLED also known as a Light-Emitting Polymer (LEP). Asmall-molecule OLED that is deposited on a substrate can be used. AnOLED that is deposited on a surface by vapor-deposition technique can beused. An OLED can be deposited on a surface by, for example,silk-screening. An LEP can be used that is deposited by, for example,solvent coating.

According to various embodiments, an OLED is used and can be formed fromone or more stable, organic materials. The OLED can include one or morecarbon-based thin films and the OLED can be capable of emitting light ofvarious colors when a voltage is applied across the one or morecarbon-based thin films. Various LEDs can use different films, forexample, quantum dot based LEDs, can use Indium tin oxide.

According to various embodiments, the OLED can include a film that islocated between two electrodes. The electrodes can be, for example, atransparent anode, a metallic cathode, or combinations thereof. Severalseparate emission areas can be stimulated between a single set ofelectrodes where simultaneous illumination of the separate emissionareas is required. According to such embodiments, only one power andcontrol module might be required for several apparent light sources. TheOLED film can include one or more of a hole-injection layer, ahole-transport layer, an emissive layer, and an electron-transportlayer. The OLED can include a film that is about one micrometer inthickness, or less. When an appropriate voltage is applied to the film,the injected positive and negative charges can recombine in the emissivelayer to produce light by means of electroluminescence. The amount oflight emitted by the OLED can be related to the voltage applied throughthe electrodes to the thin film of the OLED. Various materials suitablefor fabrication of OLEDs are available, for example, from H. W. SandsCorp. of Jupiter, Flo. Various types of OLEDs are described, forexample, in U.S. Pat. No. 4,356,429 to Tang, U.S. Pat. No. 5,554,450 toShi et al., and U.S. Pat. No. 5,593,788 to Shi et al., all of which areincorporated herein in their entireties by reference.

According to various embodiments, an OLED can be used and/or produced ona flexible substrate, on an optically clear substrate, on a substrate ofan unusual shape, or on a combination thereof. Multiple OLEDs can becombined on a substrate, wherein the multiple OLEDs can emit light atdifferent wavelengths. Multiple OLEDs on a single substrate or multipleadjacent substrates can form an interlaced or a non-interlaced patternof light of various wavelengths. The pattern can correspond to, forexample, a sample reservoir arrangement. One or more OLEDs can form ashape surrounding, for example, a sample reservoir, a series of samplereservoirs, an array of a plurality of sample reservoirs, or a sampleflow path. The sample path can be, for example, a channel, a capillary,or a micro-capillary. One or more OLEDs can be formed to follow thesample flow path. One or more OLEDs can be formed in the shape of asubstrate or a portion of a substrate. For example, the OLED can becurved, circular, oval, rectangular, square, triangular, annular, or anyother geometrically regular shape. The OLED can be formed as anirregular geometric shape. The OLED can illuminate one or more samplereservoirs, for example, an OLED can illuminate one, two, three, four,or more sample reservoirs simultaneously, or in sequence. The OLED canbe designed, for example, to illuminate all the wells of a correspondingmulti-well array.

According to various embodiments, one or more excitation filters can beincorporated into the OLED substrate, thus eliminating additionalequipment and reducing the amount of space needed for an optical system.For example, one or more filters can be formed in a layer of a substrateincluding one or more OLEDs and a layer including a sample flow path.The wavelength emitted by the OLED can be tuned by printing afluorescent dye in the OLED substrate, as taught, for example, by Hebneret al. in “Local Tuning of Organic Light-Emitting Diode Color by DyeDroplet Application,” APPLIED PHYSICS LETTERS, Vol. 73, No. 13 (Sept.28, 1998), which is incorporated herein by reference in its entirety.When using multiple emission lines in an OLED, the OLED can be used incombination with a multiple notch emission filter.

According to various embodiments, an OLED can be substituted for an LEDin any of the devices, systems, apparatuses, or methods describedherein, wherein an LED is included or used. The OLED light source canhave several OLED films stacked and operatively disposed such thatseveral wavelengths of excitation beams can traverse the same opticalpath to illuminate the sample well. Several OLEDs forming excitationbeams of the same wavelength can be stacked to provide higher output toilluminate the sample well.

According to various embodiments, an ELED can be substituted for an LEDin any of the systems, devices, apparatuses, or methods describedherein, wherein an LED is included or used. The ELED light source can bea light-emitting diode with output that emanates from betweenheterogeneous layers. An ELED can have greater radiance and couplingefficiency to an optical fiber or integrated optical circuit than asurface-emitting LED.

According to various embodiments, the light source can be a Solid StateLaser (SSL) or a micro-wire laser. The SSL can produce monochromatic,coherent, directional light and can provide a narrow wavelength ofexcitation energy. The SSL can use a lasing material that is distributedin a solid matrix, in contrast to other lasers that use a gas, dye, orsemiconductor for the lasing source material. Examples of solid statelasing materials and corresponding emission wavelengths can include, forexample: Ruby at about 694 nm; Nd:Yag at about 1064 nm; Nd:YVO4 at about1064 nm and/or about 1340 nm and which can be doubled to emit at about532 nm or about 670 nm; Alexandrite at from about 655 nm to about 815nm; and Ti: Sapphire at from about 840 nm to about 1100 nm. Micro-wirelasers are lasers where the wavelength of an excitation beam formed bythe laser can be tuned or adjusted by altering the size of a wire.According to various embodiments, other solid state lasers known tothose skilled in the art can also be used, for example, laser diodes.The appropriate lasing material can be selected based on the fluorescingdyes used, the excitation wavelength required, or both.

According to various embodiments, a Vertical Cavity Laser (VCL) can beused as an excitation source. A VCL can be substituted for an LED in anyof the devices, systems, apparatuses, or methods described herein,wherein an LED is included or used. A VCL can be a type ofsurface-emitting laser diode that uses dielectric mirrors to producesurface emission. The laser cavity can be established in a verticaldirection with respect to the plane of the active region. Examples,uses, and descriptions of VCL's can be found in U.S. Pat. No. 4,999,842,for example, that is incorporated herein in its entirety by reference.

If a SSL is used, the laser can be selected to closely match theexcitation wavelength of a fluorescent dye. The operating temperature ofthe system can be considered in selecting an appropriate SSL. Theoperating temperature can be regulated or controlled to change theemitted wavelength of the SSL. The light source for the laser can be anysource as known to those skilled in the art, such as, for example, aflash lamp. Useful information about various solid state lasers can befound at, for example, www.repairfaq.org/sam/lasersl.htm. Examples ofsolid state lasers used in various systems for identification ofbiological materials can be found in, for example, U.S. Pat. No.5,863,502 to Southgate et al. and U.S. Pat. No. 6,529,275 B2 toAmirkhanian et al.; both of which are incorporated herein by referencein their entireties.

According to various embodiments, various types of light sources can beused singularly or in combination with other light sources. One or moreOLEDs can be used with, for example, one or more non-organic LEDs, oneor more solid state lasers, one or more halogen light sources, orcombinations thereof.

According to various embodiments, an OLED layout can be connected to thepower supply through leads arranged at opposite comers of the OLEDlayout. The power supply can include or be connected to one or more of aswitch, a meter, an oscillator, a potentiometer, a detector, a signalprocessing unit, or the like. The OLED layout can include a plurality ofindividually addressable OLED lighting elements (not shown) with aseparate lead connected to each lighting element. The wiring, leads,terminals, connection arms, and the like can be implemented in, forexample, a substrate or a film. The OLED layout can be shaped to bealigned with, for example, a plurality of detection zones. Otherembodiments of OLED layouts using various shapes and various numbers ofwell lamps are within the scope of the present teachings.

According to various embodiments, each individually addressable OLEDlighting elements can include, for example, four individual lamps orOLED layers, capable of producing excitation wavelengths at fourdifferent frequencies.

The OLED layout can be constructed of a unitary or multi-partconstruction, of molded material, of stamped material, of screen printedmaterial, of cut material, or the like.

FIG. 14 illustrates an exemplary embodiment of a light source layout. AnOLED layout 450 can include varying color OLEDs 452, 454, and 456stacked upon each other. The layout can be useful for a compact lightsource design capable of forming excitation beams at varyingwavelengths. The OLEDs 452, 454, and 456 can be transparent, allowingexcitation beams from each OLED to pass through any other OLED so as tobe directed towards a sample. The OLEDs 452, 454, and 456 can emitdifferent colors, same colors, or a combination thereof depending on thecolor intensity and variety required. The OLEDs 452, 454, and 456 canshare an electrode, for example, a cathode. One electrode, for example,an anode, for powering each of the OLEDs 452, 454, and 456 can beconnected in electrical isolation from each respective anode to acontrol unit (not shown) if the capability to independently activateeach of the OLEDs 452, 454, and 456 is desired. The OLEDs 452, 454, and456 can electrically share one electrode, two electrodes, or noelectrodes. Any number of OLEDs can be stacked, for example, two OLEDs,three OLEDs, four OLEDs, or more OLEDs, to form a light source, arespective light source, or an array of light sources.

According to various embodiments, multiple excitation wavelengths can beused to detect multiple sample components. According to variousembodiments, the apparatus and method can be adapted for use by anysuitable fluorescence detection system. For example, various embodimentsof the apparatus and method can be used in a sequencing system withsingle or multiple samples, for example, in a nucleic acid sequenceamplification reaction, in a sequencing detection system.

Various embodiments of the teachings are described herein. The teachingsare not limited to the specific embodiments described, but encompassequivalent features and methods as known to one of ordinary skill in theart. Other embodiments will be apparent to those skilled in the art fromconsideration of the present specification and practice of the teachingsdisclosed herein. It is intended that the present specification andexamples be considered as exemplary only.

1. An apparatus for detecting analytes in a sample containing at leastone analyte, the apparatus comprising: a flow cytometry system includinga channel having one or more detection zones; one or more irradiationsources disposed for irradiating the one or more detection zones withnon-coherent radiation; at least one detector disposed for collecting atleast one charge corresponding to an emission beam emitted from the oneor more detection zones, the at least one detector having at least oneoutput; modulating optics disposed between the irradiation source andthe at least one detector; and a time delay integration system coupledto the at least one detector for effecting time delay integration of theat least one charge by accumulating the at least one charge beforereading the at least one charge at the output of the at least onedetector, wherein the time delay integration system accumulates the atleast one charge by moving, relative to one another, the modulatingoptics and the channel.
 2. The apparatus of claim 1, wherein the flowcytometry system comprises an electric field gradient source.
 3. Theapparatus of claim 1, wherein the one or more irradiation sourcescomprises one or more light emitting diodes.
 4. The apparatus of claim3, wherein the one or more light emitting diodes comprises one or moreorganic light emitting diodes.
 5. The apparatus of claim 1, wherein themodulating optics includes a relay lens system comprising a collimatinglens and a re-imaging lens.
 6. The apparatus of claim 5, wherein themodulating optics includes a conditioning filter disposed between theone or more irradiation sources and the one or more detection zones. 7.The apparatus of claim 6, wherein the conditioning filter comprises alongpass filter, a shortpass filter, a multi-notch filter, abeamsplitter, or a combination thereof.
 8. The apparatus of claim 6,wherein the modulating optics includes a focusing lens disposed betweenthe conditioning filter and the one or more detection zones.
 9. Theapparatus of claim 5, wherein the modulating optics includes atransmission grating disposed between the focusing lens and there-imaging lens.
 10. The apparatus of claim 5, wherein the modulatingoptics includes a filter disposed between the one or more detectionzones and the at least one detector for filtering through only theemission beam.
 11. The apparatus of claim 10, wherein the filtercomprises a longpass filter, a shortpass filter, a multi-notch filter, abandpass filter, a beam splitter, or a combination thereof.
 12. Theapparatus of claim 1, further comprising a sample containing wholecells.
 13. The apparatus of claim 1, further comprising a samplecontaining the at least one analyte labeled with at least one marker.14. The apparatus of claim 13, wherein the at least one marker comprisesa dye marker, a fluorescing dye, a free-floating dye, a reporter dye, aprobe dye, an intercalating dye, a quantum dot, a molecular beacon, aquantum dot media, a quantum dot bead, a dye-labeled bead, a dyeattached to an analyte assoicated with a bead, or a combination thereof.15. The apparatus of claim 13, wherein the modulating optics comprise atleast one conditioning filter for each irradiation source of the one ormore irradiation sources, each respective conditioning filter beingeffective for substantially blocking predetermined excitationwavelengths to produce conditioned light, wherein the predeterminedexcitation wavelengths of each marker do not conflict with theexcitation spectra of each other marker.
 16. The apparatus of claim 13,wherein the modulating optics comprises at least one long pass filterand at least one bandpass filter for each irradiation source of the oneor more irradiation sources, each of the at least one long pass filterand each of the at least one bandpass filter being effective for lettingthrough, substantially exclusively, predetermined wavelengths of lightfrom the one or more detection zones corresponding to a portion of thewavelengths of the light signals emitted by the at least one marker, tothereby produce filtered light.
 17. The apparatus of claim 1, furthercomprising a separating device.
 18. The apparatus of claim 17, whereinthe seperating device includes a plurality of separation regions. 19.The apparatus of claim 1, wherein the one or more irradiation sourcescomprises a single light emitting diode, and the apparatus furthercomprises a device capable of spectrally distributing the light signalsto thereby produce spectrally distributed light.
 20. The apparatus ofclaim 16, wherein the one or more irradiation sources comprises aplurality of light emitting diodes each emitting light in a respectivepredetermined frequency range, and the respective bandpass filtercomprises a plurality of bandpass filters each associated with arespective one of the plurality of light emitting diodes, eachrespective bandpass filter being effective for letting through,substantially exclusively, predetermined wavelengths of light from theone or more detection zones corresponding to a portion of thewavelengths of the light signals emitted by each marker of the at leastone marker to thereby produce filtered light.
 21. The apparatus of claim15, further comprising an offset system for spatially offsetting, on theat least one detector, at least one image for each conditioning filterby a predetermined amount.
 22. The apparatus of claim 21, wherein theoffset system comprises a plurality of offset mechanisms, eachassociated with a respective one of the conditioning filters.
 23. Theapparatus of claim 22, wherein each offset mechanism includes one ormore of a glass plate, a grating, a mirror, or a combination thereof.24. The apparatus of claim 21, wherein the offset system is adapted toeffect a translational movement of at least one of the at least onedetector, the modulating optics, and the one or more detection zones,with respect to one another, for spatially offsetting the at least oneimage by a second predetermined amount.
 25. The apparatus of claim 15,further comprising a filter wheel, the bandpass filters being disposedon the filter wheel, each respective bandpass filter further beingselectively positionable with respect to the one or more detection zonesfor filtering light emitted from the one or more detection zones by theat least one marker associated with each respective bandpass filter. 26.The apparatus of claim 15, wherein the time delay integration system iscapable of controlling the at least one detector to read the at leastone accumulated charge on a frame by frame basis, each framecorresponding to the at least one accumulated charge on the at least onedetector during an integration time and produced by the conditionedlight through each conditioning filter.
 27. The apparatus of claim 1,wherein the time delay integration system is capable of controlling theat least one detector to read the at least one accumulated charge on acontinuous basis.
 28. The apparatus of claim 1, wherein the at least onedetector comprises a two-dimensional charge-coupled device.
 29. Theapparatus of claim 1, wherein: the one or more irradiation sourcesincludes a plurality of light emitting diodes adapted to simultaneouslyirradiate the one or more detection zones, each of the light emittingdiodes illuminating a separate one of the one or more detection zones;and the apparatus further comprises masks to selectively mask thechannel such that the light signals from the respective one or moredetection zones are distinct.
 30. The apparatus of claim 12, wherein thetime delay integration system includes a system coupled to themodulating optics for moving the modulating optics relative to thechannel at a speed that is synchronized to a movement of the at leastone analyte across the one or more detection zones.
 31. The apparatus ofclaim 12, wherein the time delay integration system comprises a systemcoupled to the channel for moving the channel relative to the modulatingoptics at a speed that is synchronized to a movement of the at least oneanalyte across the one or more detection zones.
 32. The apparatus ofclaim 12, further including a sorting system including one or morecollection channels and a controller for controlling a motive force foreach collection channel, wherein the sorting system is capable ofsorting each analyte into a respective one of the one or more collectionchannels.
 33. The apparatus of claim 32, wherein the motive forcecomprises an electrokinetic force, a mechanical force, a production ofelectric field gradient, a switchable electric field, vacuum, a streamof air, or a combination thereof.
 34. The apparatus of claim 32, whereinthe controller comprises more than one controller, each controllerassociated with a respective collection channel.
 35. An apparatus fordetecting analytes in a sample, comprising: a flow cytometry systemincluding a channel having at least one detection zone; one or moreirradiation sources disposed for irradiating the at least one detectionzone with radiation, wherein the one or more irradiation sourcesincludes a solid state laser or a micro-wire laser; at least onedetector disposed for collecting at least one charge corresponding to anemission beam emitted from the at least one detection zone, eachdetector of the at least one detector having an output; modulatingoptics disposed between the irradiation source and the at least onedetector; and a time delay integration system coupled to the at leastone detector for effecting time delay integration of the at least onecharge by accumulating the at least one charge before reading the atleast one charge at the output of the at least one detector, wherein thetime delay integration system accumulates the at least one charge bymoving, relative to one another, the modulating optics and the channel.36. The apparatus of claim 35, wherein the one or more irradiationsources comprises one or more solid state laser sources.
 37. Theapparatus of claim 35, wherein the one or more irradiation sourcescomprises one or more micro-wire laser sources.
 38. A method fordetecting and sorting analytes, comprising: providing a flow cytometrysystem comprising a channel including at least one detection zone;providing a sample containing at least one detectable analyte;irradiating the at least one detection zone using one or moreirradiation sources disposed for irradiating the at lease one detectionzone with radiation so that the at least one detectable analyte isresponsive to the radiation and produces emission beams indicative ofthe at least one detectable analyte; detecting the emission beams withat least one detector to produce at least one charge on the at least onedetector, corresponding to the detected emission beams; modulating lightbetween the one or more irradiation sources and the at least onedetector using modulating optics; effecting a time delay integration ofat least one charge by accumulating the at least one charge associatedwith the at least one detectable analyte during an integration time ofthe at least one detectable analyte before reading the at least onecharge at an output of the at least one detector, wherein theaccumulation is effected by moving, relative to one another, themodulating optics and the channel; reading the accumulated charges; andsorting the at least one analyte based on the read accumulated charges.39. The method of claim 38, further comprising separating the at leastone detectable analyte.
 40. The method of claim 38, wherein the step ofmodulating comprises: substantially blocking predetermined wavelengthsof light emitted by a respective irradiation source of the one or moreirradiation sources to thereby produce conditioned light, thepredetermined wavelengths being those wavelengths not overlapping theemission spectra each of the at least one marker responsive to therespective one of the one or more irradiation sources; focusing theconditioned light onto the at least one detection zone; collimatinglight from the at least one detection zone to thereby generatecollimated light; and filtering the light from the at least onedetection zone by letting through, substantially exclusively,predetermined wavelengths of the light signals from the at least onedetection zone corresponding to a portion of the wavelengths of thelight signals emitted by the at least one marker, to thereby producefiltered light.
 41. The method of claim 40, further comprising the stepof focusing the filtered light onto the at least one detector.
 42. Themethod of claim 38, wherein the one or more irradiation sources compriseat least one of a light emitting diode, an organic light emitting diode,a quantum dot, a solid state laser, a micro-wire laser.
 43. The methodof claim 38, wherein said method further comprises the step of providinga plurality of bandpass filters each associated with a respective one ofthe irradiation sources, each respective bandpass filter being effectivefor filtering light from the at least one detection zone by lettingthrough, substantially exclusively, predetermined wavelengths of thelight from the at least one detection zone corresponding to a portion ofthe wavelengths of the light signals emitted by each of the at least onemarker, to thereby produce filtered light.
 44. The method of claim 43,further comprising the step of spatially offsetting the filtered lightfrom each respective bandpass filter by a predetermined amount as afunction of said each respective bandpass filter such that at least oneimage on the at least one detector is produced by the at least onecharge spatially offset from one another.
 45. An apparatus for sortinganalytes in a sample containing at least one detectable analyte, theapparatus comprising: a channel having one or more detection zones; oneor more irradiation sources disposed for irradiating the one or moredetection zones with non-coherent radiation; at least one detectordisposed for collecting light signals emitted from the at least onedetectable analyte in the one or more detection zones excited by theradiation, the at least one detector having an output; a time delayintegration system coupled to the at least one detector for effectingthe time delay integration of at least one charge on the at least onedetector, corresponding to the light signals by accumulating the atleast one charge before reading the at least one charge at the output ofthe at least one detector; and a sorting system capable of directing theflow of at least one detectable analyte.
 46. The apparatus of claim 45,wherein the sorting system comprises a controller in communication withthe at least one detector, wherein the controller is capable ofcontrolling a motive force to effect sorting a detectable analyte. 47.The apparatus of claim 46, wherein the motive force can be selected froman electrokinetic force, a mechanical force, an electric field gradient,a switchable electric field, a vacuum, a stream of air, or a combinationthereof.
 48. The apparatus of claim 47, wherein the motive force is amechanical force, and the apparatus further comprises at least onecollection tube and a mechanical arm, wherein the mechanical arm isconnected to the collection tube and electrically connected to thecontroller.
 49. A method for sorting analytes in a sample containing atleast one detectable analyte, comprising: providing achannel-defining-member defining a channel therein having at least onedetection zone; separating a sample containing at least one detectableanalyte moving through the channel; irradiating the at least detectionzone using one or more irradiation sources generating radiation of suchwavelength as to thereby excite the at least one detectable analyte andcause the at least one detectable analyte to emit light signalsindicative of the at least one detectable analyte; detecting the lightsignals produced by the at least one detectable analyte by collectingthe light signals on at least one detector to produce charges on the atleast one detector corresponding to the light signals; modulating lightbetween the one or more irradiation sources and the at least onedetector using modulating optics; effecting a time delay integration ofthe light signals within the at least one detector by accumulating atleast one charge within the at least one detector corresponding to lightsignals associated with the at least one detectable analyte during anintegration time of the at least one detectable analyte moving acrossthe at least one detection zone, wherein the accumulation is effected bymoving, relative to one another, the modulating optics and the channel;reading the at least one accumulated charge; and sorting a detectableanalyte of the sample based on the reading of the at least oneaccumulated charge.
 50. The method of claim 49, wherein sorting thesample comprises administering a motive force to one or more analytes ofthe sample to control movement of the one or more analytes.
 51. Themethod of claim 50, wherein the motive force is an electrokinetic force,a switchable electric field, a mechanical force, an electrical fieldgradient, a vacuum, a stream of air, or a combination thereof.